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Zhylkibayev A, Mobley J, Athar M, Gorbatyuk M. A multiomic study of retinal tissues in mice with direct ocular exposure to vesicants. Exp Eye Res 2025; 257:110414. [PMID: 40379201 DOI: 10.1016/j.exer.2025.110414] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2025] [Revised: 04/03/2025] [Accepted: 05/01/2025] [Indexed: 05/19/2025]
Abstract
This study employed a multiomic approach to investigate retinal tissue damage following direct ocular exposure (DOE) to vesicants (VSs)-namely, nitrogen mustard (NM) and lewisite (Lew). We explored both the acute and chronic stages of retinal injury by assessing functional, structural, and molecular changes. C57BL/6 mice were used to measure scotopic and photopic electroretinograms (ERGs) and to analyze TUNEL-positive retinal cells. Global retinal proteomics was conducted to identify common and unique signaling pathways. In addition, we performed targeted metabolomic and lipidomic analyses of retinal tissue to uncover significant metabolic changes. Our results demonstrated remarkable declines in ERG amplitudes at 2 and 4 weeks post-exposure, accompanied by an increase in TUNEL+ retinal cells in response to DOE to both VSs. Our proteomic analysis revealed chronic oxidative stress, mitochondrial dysfunction, elevated RXR signaling, and increased levels of 28 proteins. Moreover, we observed a decline in the KEGG phototransduction pathways, along with the downregulation of photoreceptor-specific proteins, in response to both VSs. Consistent with the proteomic findings, targeted metabolomics identified a decline in phototransduction and steroid hormone biosynthesis, along with increases in D-amino acid and purine metabolism, as well as lysine degradation. These changes were associated with a GSSG/GSH ratio of 2.6, confirming the proteomic data on oxidative stress. Furthermore, lipidomic analysis revealed an increase in oxidative lipid levels, accompanied by a 3.4-fold increase in phosphatidylserine (PS), suggesting apoptotic cell death and a reduction in fatty acids (FAs). In conclusion, exposure to both VSs induced progressive retinal damage, altering major metabolic pathways and dysregulating lipid metabolism. Future studies should focus on identifying the responses of individual neuronal cell types to DOE to VSs to develop cell-specific countermeasures.
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Affiliation(s)
- Assylbek Zhylkibayev
- Wake Forest University, School of Medicine, Department of Biochemistry, Winston-Salem, NC, USA.
| | - James Mobley
- University of Alabama at Birmingham, School of Medicine, Department of Anesthesiology and Perioperative Medicine, Birmingham, AL, USA.
| | - Mohammad Athar
- University of Alabama at Birmingham, School of Medicine, Department of Dermatology, Birmingham, AL, USA.
| | - Marina Gorbatyuk
- Wake Forest University, School of Medicine, Department of Biochemistry, Winston-Salem, USA.
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2
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Tan K, Sebat J, Wilkinson M. Cell type- and factor-specific nonsense-mediated RNA decay. Nucleic Acids Res 2025; 53:gkaf395. [PMID: 40366162 PMCID: PMC12076418 DOI: 10.1093/nar/gkaf395] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2024] [Revised: 04/17/2025] [Accepted: 04/28/2025] [Indexed: 05/15/2025] Open
Abstract
Nonsense-mediated RNA decay (NMD) is a highly conserved RNA turnover pathway that influences several biological processes. Specific features in messenger RNAs (mRNAs) have been found to trigger decay by NMD, leading to the assumption that NMD sensitivity is an intrinsic quality of a given transcript. Here, we provide evidence that, instead, an overriding factor dictating NMD sensitivity is the cell environment. Using several genome-wide techniques to detect NMD-target mRNAs, we find that hundreds of mRNAs are sensitized to NMD as human embryonic stem cells progress to form neural progenitor cells. Another class of mRNAs escape from NMD during this developmental progression. We show that the differential sensitivity to NMD extends to in vivo scenarios, and that the RNA-binding protein, HNRNPL, has a role in cell type-specific NMD. We also addressed another issue in the field-whether NMD factors are core or branch-specific in their action. Surprisingly, we found that UPF3B, an NMD factor critical for the nervous system, shares only 30% of NMD-target transcripts with the core NMD factor UPF2. Together, our findings have implications for how NMD is defined and measured, how NMD acts in different biological contexts, and how different NMD branches influence human diseases.
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Affiliation(s)
- Kun Tan
- Department of Obstetrics, Gynecology, and Reproductive Sciences, School of Medicine, University of California San Diego, La Jolla, CA 92093, United States
| | - Jonathan Sebat
- Department of Psychiatry, Department of Cellular and Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA 92093, United States
- Institute of Genomic Medicine, University of California San Diego, La Jolla, CA 92093, United States
| | - Miles F Wilkinson
- Department of Obstetrics, Gynecology, and Reproductive Sciences, School of Medicine, University of California San Diego, La Jolla, CA 92093, United States
- Institute of Genomic Medicine, University of California San Diego, La Jolla, CA 92093, United States
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3
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Cano-Besquet S, Park M, Berkley N, Wong M, Ashiqueali S, Noureddine S, Gesing A, Schneider A, Mason J, Masternak MM, Dhahbi JM. Gene and transcript expression patterns, coupled with isoform switching and long non-coding RNA dynamics in adipose tissue, underlie the longevity of Ames dwarf mice. GeroScience 2025; 47:1923-1943. [PMID: 39405012 PMCID: PMC11978586 DOI: 10.1007/s11357-024-01383-x] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2024] [Accepted: 10/06/2024] [Indexed: 04/09/2025] Open
Abstract
Our study investigates gene expression in adipose tissue of Ames dwarf (df/df) mice, whose deficiency in growth hormone is linked to health and extended lifespan. Recognizing adipose tissue influence on metabolism, aging, and related diseases, we aim to understand its contribution to the health and longevity of df/df mice. We have identified gene and transcript expression patterns associated with critical biological functions, including metabolism, stress response, and resistance to cancer. Intriguingly, we identified genes that, despite maintaining unchanged expression levels, switch between different isoforms, impacting essential cellular functions such as tumor suppression, oncogenic activity, ATP transport, and lipid biosynthesis and storage. The isoform switching is associated with changes in protein domains, retention of introns, initiation of nonsense-mediated decay, and emergence of intrinsically disordered regions. Moreover, we detected various alternative splicing events that may drive these structural alterations. We also found changes in the expression of long non-coding RNAs (lncRNAs) that may be involved in the aging process and disease resistance by regulating crucial genes in survival and metabolism. Through weighted gene co-expression network analysis, we have linked four lncRNAs with 29 genes, which contribute to protein complexes such as the Mili-Tdrd1-Tdrd12 complex. Beyond safeguarding DNA integrity, this complex also has a wider impact on gene regulation, chromatin structure, and metabolic control. Our detailed investigation provides insight into the molecular foundations of the remarkable health and longevity of df/df mice, emphasizing the significance of adipose tissue in aging and identifying new avenues for health-promoting therapeutic strategies.
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Affiliation(s)
- Sebastian Cano-Besquet
- Department of Medical Education, School of Medicine, California University of Science & Medicine, Colton, CA, USA
| | - Maiyon Park
- Department of Medical Education, School of Medicine, California University of Science & Medicine, Colton, CA, USA
| | | | - Michelle Wong
- Department of Medical Education, School of Medicine, California University of Science & Medicine, Colton, CA, USA
| | - Sarah Ashiqueali
- College of Medicine, Burnett School of Biomedical Sciences, University of Central Florida, Orlando, FL, USA
| | - Sarah Noureddine
- College of Medicine, Burnett School of Biomedical Sciences, University of Central Florida, Orlando, FL, USA
| | - Adam Gesing
- Department of Endocrinology of Ageing, Medical University of Lodz, Lodz, Poland
| | - Augusto Schneider
- Faculdade de Nutrição, Universidade Federal de Pelotas, Pelotas, Brazil
| | - Jeffrey Mason
- College of Veterinary Medicine, Department of Veterinary Clinical and Life Sciences, Center for Integrated BioSystems, Utah State University, Logan, UT, USA
| | - Michal M Masternak
- College of Medicine, Burnett School of Biomedical Sciences, University of Central Florida, Orlando, FL, USA
- Department of Head and Neck Surgery, Poznan University of Medical Sciences, Poznan, Poland
| | - Joseph M Dhahbi
- Department of Medical Education, School of Medicine, California University of Science & Medicine, Colton, CA, USA.
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4
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Flores-Mendez M, Tintos-Hernández JA, Ramos-Rodriguez L, Miles L, Lo TY, Song Y, Ortiz-González XR. TBCK-deficiency leads to compartment-specific mRNA and lysosomal trafficking defects in patient-derived neurons. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.03.02.641041. [PMID: 40093117 PMCID: PMC11908138 DOI: 10.1101/2025.03.02.641041] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 03/19/2025]
Abstract
Monogenic pediatric neurodegenerative disorders can reveal fundamental cellular mechanisms that underlie selective neuronal vulnerability. TBCK-Encephaloneuronopathy (TBCKE) is a rare autosomal recessive disorder caused by stop-gain variants in the TBCK gene. Clinically, patients show evidence of profound neurodevelopmental delays, but also symptoms of progressive encephalopathy and motor neuron disease. Yet, the physiological role of TBCK protein remains unclear. We report a human neuronal TBCKE model, derived from iPSCs homozygous for the Boricua variant (p.R126X). Using unbiased proteomic analyses of human neurons, we find TBCK interacts with PPP1R21, C12orf4, and Cryzl1, consistent with TBCK being part of the FERRY mRNA transport complex. Loss of TBCK leads to depletion of C12ORF4 protein levels across multiple cell types, suggesting TBCK may also play a role regulating at least some members of the FERRY complex. We find that TBCK preferentially, but not exclusively, localizes to the surface of endolysosomal vesicles and can colocalize with mRNA in lysosomes. Furthermore, TBCK-deficient neurons have reduced mRNA content in the axonal compartment relative to the soma. TBCK-deficient neurons show reduced levels of the lysosomal dynein/dynactin adapter protein JIP4, which functionally leads to TBCK-deficient neurons exhibiting striking lysosomal axonal retrograde trafficking defects. Hence, our work reveals that TBCK can mediate endolysosomal trafficking of mRNA, particularly along lysosomes in human axonal compartments. TBCK-deficiency leads to compartment-specific mRNA and lysosomal trafficking defects in neurons, which likely contribute to the preferential susceptibility to neurodegeneration.
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Affiliation(s)
- Marco Flores-Mendez
- Department of Pediatrics, Division of Neurology, The Children's of Philadelphia, Philadelphia, PA
- Center for Mitochondrial and Epigenomic Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA
| | - Jesus A Tintos-Hernández
- Department of Pediatrics, Division of Neurology, The Children's of Philadelphia, Philadelphia, PA
- Center for Mitochondrial and Epigenomic Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA
| | - Leonardo Ramos-Rodriguez
- Department of Biomedical Graduate Studies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
| | - Leann Miles
- Department of Biomedical Graduate Studies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
| | - Tsz Y Lo
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA
| | - Yuanquan Song
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA
- Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA
| | - Xilma R Ortiz-González
- Department of Pediatrics, Division of Neurology, The Children's of Philadelphia, Philadelphia, PA
- Center for Mitochondrial and Epigenomic Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA
- Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
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5
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Dowdle ME, Lykke-Andersen J. Cytoplasmic mRNA decay and quality control machineries in eukaryotes. Nat Rev Genet 2025:10.1038/s41576-024-00810-1. [PMID: 39870755 DOI: 10.1038/s41576-024-00810-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/09/2024] [Indexed: 01/29/2025]
Abstract
mRNA degradation pathways have key regulatory roles in gene expression. The intrinsic stability of mRNAs in the cytoplasm of eukaryotic cells varies widely in a gene- and isoform-dependent manner and can be regulated by cellular cues, such as kinase signalling, to control mRNA levels and spatiotemporal dynamics of gene expression. Moreover, specialized quality control pathways exist to rid cells of non-functional mRNAs produced by errors in mRNA processing or mRNA damage that negatively impact translation. Recent advances in structural, single-molecule and genome-wide methods have provided new insights into the central machineries that carry out mRNA turnover, the mechanisms by which mRNAs are targeted for degradation and the general principles that govern mRNA stability at a global level. This improved understanding of mRNA degradation in the cytoplasm of eukaryotic cells is finding practical applications in the design of therapeutic mRNAs.
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Affiliation(s)
- Megan E Dowdle
- Department of Molecular Biology, School of Biological Sciences, University of California San Diego, La Jolla, CA, USA
| | - Jens Lykke-Andersen
- Department of Molecular Biology, School of Biological Sciences, University of California San Diego, La Jolla, CA, USA.
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6
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Jeon S, Jeon Y, Lim JY, Kim Y, Cha B, Kim W. Emerging regulatory mechanisms and functions of biomolecular condensates: implications for therapeutic targets. Signal Transduct Target Ther 2025; 10:4. [PMID: 39757214 DOI: 10.1038/s41392-024-02070-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2024] [Revised: 10/01/2024] [Accepted: 11/06/2024] [Indexed: 01/07/2025] Open
Abstract
Cells orchestrate their processes through complex interactions, precisely organizing biomolecules in space and time. Recent discoveries have highlighted the crucial role of biomolecular condensates-membrane-less assemblies formed through the condensation of proteins, nucleic acids, and other molecules-in driving efficient and dynamic cellular processes. These condensates are integral to various physiological functions, such as gene expression and intracellular signal transduction, enabling rapid and finely tuned cellular responses. Their ability to regulate cellular signaling pathways is particularly significant, as it requires a careful balance between flexibility and precision. Disruption of this balance can lead to pathological conditions, including neurodegenerative diseases, cancer, and viral infections. Consequently, biomolecular condensates have emerged as promising therapeutic targets, with the potential to offer novel approaches to disease treatment. In this review, we present the recent insights into the regulatory mechanisms by which biomolecular condensates influence intracellular signaling pathways, their roles in health and disease, and potential strategies for modulating condensate dynamics as a therapeutic approach. Understanding these emerging principles may provide valuable directions for developing effective treatments targeting the aberrant behavior of biomolecular condensates in various diseases.
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Affiliation(s)
- Soyoung Jeon
- Department of Life Science, University of Seoul, Seoul, South Korea
| | - Yeram Jeon
- Department of Life Science, University of Seoul, Seoul, South Korea
| | - Ji-Youn Lim
- New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, South Korea
| | - Yujeong Kim
- Department of Life Science, University of Seoul, Seoul, South Korea
| | - Boksik Cha
- New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, South Korea.
| | - Wantae Kim
- Department of Life Science, University of Seoul, Seoul, South Korea.
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7
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Ottens F, Efstathiou S, Hoppe T. Cutting through the stress: RNA decay pathways at the endoplasmic reticulum. Trends Cell Biol 2024; 34:1056-1068. [PMID: 38008608 DOI: 10.1016/j.tcb.2023.11.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2023] [Revised: 11/07/2023] [Accepted: 11/08/2023] [Indexed: 11/28/2023]
Abstract
The endoplasmic reticulum (ER) is central to the processing of luminal, transmembrane, and secretory proteins, and maintaining a functional ER is essential for organismal physiology and health. Increased protein-folding load on the ER causes ER stress, which activates quality control mechanisms to restore ER function and protein homeostasis. Beyond protein quality control, mRNA decay pathways have emerged as potent ER fidelity regulators, but their mechanistic roles in ER quality control and their interrelationships remain incompletely understood. Herein, we review ER-associated RNA decay pathways - including regulated inositol-requiring enzyme 1α (IRE1α)-dependent mRNA decay (RIDD), nonsense-mediated mRNA decay (NMD), and Argonaute-dependent RNA silencing - in ER homeostasis, and highlight the intricate coordination of ER-targeted RNA and protein decay mechanisms and their association with antiviral defense.
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Affiliation(s)
- Franziska Ottens
- Institute for Genetics, University of Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
| | - Sotirios Efstathiou
- Institute for Genetics, University of Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
| | - Thorsten Hoppe
- Institute for Genetics, University of Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital of Cologne, Cologne, Germany.
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8
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Li D, Al-Dahleh K, Murphy DA, Georgieva S, Matthews N, Shovlin CL. Endogenous plasma resuspension of peripheral blood mononuclear cells prevents preparative-associated stress that modifies polyA-enriched RNA responses to subsequent acute stressors. Cell Stress 2024; 11:112-124. [PMID: 39628848 PMCID: PMC11613960 DOI: 10.15698/cst2024.11.301] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2023] [Revised: 09/12/2024] [Accepted: 10/03/2024] [Indexed: 12/06/2024] Open
Abstract
Human peripheral blood mononuclear cells (PBMCs) are used to examine biological processes and disease, when basal variability in cellular activation and splicing is described and unexplained. Using isolation systems that maintained buffy coat cells (PBMCs, platelets) in their own plasma, poly-A enriched RNA-sequencing (RNASeq) detected 42,720 Ensembl gene IDs, including >95% of the top 100 Genotype Tissue Expression Project (GTEx)-expressed genes in lung, colon, heart, skeletal muscle and liver, and 10/17 clinically-actionable genes listed by the Pharmacogenomics Knowledgebase. Transcriptome changes were defined after 1h treatment with 32°C hypothermia (hsp70 family member change), 10 μmol/L ferric citrate that had no discernible effect, and 100 μg/mL cycloheximide leading to induction of primary response (immediate early) genes including IL1B and TNF. Same-donor PBMCs prepared conventionally using washes then resuspension in serum-supplemented media demonstrated basal upregulation of stress signalling pathway genes that masked and overlapped differential gene expression profiles after 100 µg/L cycloheximide. Plasma-resuspended PBMCs demonstrated minor transcriptome changes after 40 μmol/L ferric citrate, whereas consistent and greater magnitude changes were observed for washed/media-resuspended PBMCs. We conclude that endogenous plasma-maintained PBMCs provide a more robust platform to interrogate acute cellular perturbations triggering innate immunity, and that varying susceptibility of PBMCs to preparative stresses is an important component of experimental variability.
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Affiliation(s)
- Dongyang Li
- National Heart and Lung Institute, Imperial College LondonLondon, W12 ONNUK
- National Institute for Health Research (NIHR) Imperial Biomedical Research CentreLondon, W2 1NYUK
| | - Karina Al-Dahleh
- National Heart and Lung Institute, Imperial College LondonLondon, W12 ONNUK
- National Institute for Health Research (NIHR) Imperial Biomedical Research CentreLondon, W2 1NYUK
| | - Daniel A Murphy
- National Institute for Health Research (NIHR) Imperial Biomedical Research CentreLondon, W2 1NYUK
- Pharmacy, Imperial College Healthcare NHS TrustLondon, W12 OHSUK
| | - Sonya Georgieva
- National Heart and Lung Institute, Imperial College LondonLondon, W12 ONNUK
| | - Nik Matthews
- NIHR Imperial BRC Genomic Facility, Faculty of Medicine, Imperial College London
| | - Claire L Shovlin
- National Heart and Lung Institute, Imperial College LondonLondon, W12 ONNUK
- National Institute for Health Research (NIHR) Imperial Biomedical Research CentreLondon, W2 1NYUK
- Specialist Medicine , Imperial College Healthcare NHS TrustLondon, W12 OHSUK
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9
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Pérez-Mato M, Dopico-López A, Akkoc Y, López-Amoedo S, Correa-Paz C, Candamo-Lourido M, Iglesias-Rey R, López-Arias E, Bugallo-Casal A, da Silva-Candal A, Bravo SB, Chantada-Vázquez MDP, Arias S, Santamaría-Cadavid M, Estany-Gestal A, Zaghmi A, Gauthier MA, Gutiérrez-Fernández M, Martin A, Llop J, Rodríguez C, Almeida Á, Migliavacca M, Polo E, Pelaz B, Gozuacik D, El Yamani N, SenGupta T, Rundén-Pran E, Vivancos J, Castellanos M, Díez-Tejedor E, Sobrino T, Rabinkov A, Mirelman D, Castillo J, Campos F. Preclinical validation of human recombinant glutamate-oxaloacetate transaminase for the treatment of acute ischemic stroke. iScience 2024; 27:111108. [PMID: 39524351 PMCID: PMC11543921 DOI: 10.1016/j.isci.2024.111108] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2024] [Revised: 08/21/2024] [Accepted: 10/02/2024] [Indexed: 11/16/2024] Open
Abstract
The blood enzyme glutamate-oxaloacetate transaminase (GOT) has been postulated as an effective therapeutic to protect the brain during stroke. To demonstrate its potential clinical utility, a new human recombinant form of GOT (rGOT) was produced for medical use. We tested the pharmacokinetics and evaluated the protective efficacy of rGOT in rodent and non-human primate models that reflected clinical stroke conditions. We found that continuous intravenous administration of rGOT within the first 8 h after ischemic onset significantly reduced the infarct size in both severe (30%) and mild lesions (48%). Cerebrospinal fluid and proteomics analysis, in combination with positron emission tomography imaging, indicated that rGOT can reach the brain and induce cytoprotective autophagy and induce local protection by alleviating neuronal apoptosis. Our results suggest that rGOT can be safely used immediately in patients suspected of having a stroke. This study requires further validation in clinical stroke populations.
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Affiliation(s)
- María Pérez-Mato
- Translational Stroke Laboratory Group (TREAT), Clinical Neurosciences Research Laboratory (LINC), Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain
- Neurological Sciences and Cerebrovascular Research Laboratory, Department of Neurology, Neurology and Cerebrovascular Disease Group, Neuroscience Area of Hospital La Paz Institute for Health Research – IdiPAZ (La Paz University Hospital- Universidad Autónoma de Madrid), 28029 Madrid, Spain
| | - Antonio Dopico-López
- Translational Stroke Laboratory Group (TREAT), Clinical Neurosciences Research Laboratory (LINC), Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain
| | - Yunus Akkoc
- Koç University Research Center for Translational Medicine (KUTTAM), Istanbul 34450, Turkey
- Department of Medical Biology, Koç University School of Medicine, Istanbul 34450, Turkey
| | - Sonia López-Amoedo
- Translational Stroke Laboratory Group (TREAT), Clinical Neurosciences Research Laboratory (LINC), Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain
| | - Clara Correa-Paz
- Translational Stroke Laboratory Group (TREAT), Clinical Neurosciences Research Laboratory (LINC), Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain
| | - María Candamo-Lourido
- Translational Stroke Laboratory Group (TREAT), Clinical Neurosciences Research Laboratory (LINC), Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain
| | - Ramón Iglesias-Rey
- Neuroimaging and Biotechnology Laboratory Group (NOBEL), Clinical Neurosciences Research Laboratory (LINC), Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain
| | - Esteban López-Arias
- Translational Stroke Laboratory Group (TREAT), Clinical Neurosciences Research Laboratory (LINC), Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain
| | - Ana Bugallo-Casal
- Translational Stroke Laboratory Group (TREAT), Clinical Neurosciences Research Laboratory (LINC), Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain
| | - Andrés da Silva-Candal
- Neurology Service, University Hospital Complex of A Coruña, A Coruña Biomedical Research Institute, 15006 A Coruña, Spain
| | - Susana B. Bravo
- Proteomic Unit, Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain
| | - María del Pilar Chantada-Vázquez
- Proteomic Unit, Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain
- Research Unit, Lucus Augusti University Hospital (HULA), Servizo Galego de Saúde (SERGAS), 27002 Lugo, Spain
| | - Susana Arias
- Stroke Unit, Department of Neurology, Hospital Clínico Universitario, 15706 Santiago de Compostela, Spain
| | - María Santamaría-Cadavid
- Stroke Unit, Department of Neurology, Hospital Clínico Universitario, 15706 Santiago de Compostela, Spain
| | - Ana Estany-Gestal
- Unit of Methodology of the Research, Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain
| | - Ahlem Zaghmi
- Institut National de la Recherche Scientifique (INRS), EMT Research Center, Varennes, QC J3X 1S2, Canada
- Rheumatology Unit, Department of Medicine, Karolinska Institutet, Karolinska University Hospital, 171 77 Stockholm, Sweden
| | - Marc A. Gauthier
- Institut National de la Recherche Scientifique (INRS), EMT Research Center, Varennes, QC J3X 1S2, Canada
| | - María Gutiérrez-Fernández
- Neurological Sciences and Cerebrovascular Research Laboratory, Department of Neurology, Neurology and Cerebrovascular Disease Group, Neuroscience Area of Hospital La Paz Institute for Health Research – IdiPAZ (La Paz University Hospital- Universidad Autónoma de Madrid), 28029 Madrid, Spain
| | - Abraham Martin
- Achucarro Basque Center for Neuroscience, 48940 Leioa, Spain
- Ikerbasque Basque Foundation for Science, 48009 Bilbao, Spain
| | - Jordi Llop
- CIC biomaGUNE, Basque Research and Technology Alliance (BRTA), 20014 San Sebastian, Spain
| | - Cristina Rodríguez
- Institute of Functional Biology and Genomics (IBFG), CSIC, University of Salamanca, 37007 Salamanca, Spain
- Institute of Biomedical Research of Salamanca (IBSAL), University Hospital of Salamanca, CSIC, University of Salamanca, 37007 Salamanca, Spain
| | - Ángeles Almeida
- Institute of Functional Biology and Genomics (IBFG), CSIC, University of Salamanca, 37007 Salamanca, Spain
- Institute of Biomedical Research of Salamanca (IBSAL), University Hospital of Salamanca, CSIC, University of Salamanca, 37007 Salamanca, Spain
| | - Martina Migliavacca
- Center for Research in Biological Chemistry and Molecular Materials (CiQUS), University of Santiago de Compostela, 15705 Santiago de Compostela, Spain
| | - Ester Polo
- Center for Research in Biological Chemistry and Molecular Materials (CiQUS), University of Santiago de Compostela, 15705 Santiago de Compostela, Spain
| | - Beatriz Pelaz
- Center for Research in Biological Chemistry and Molecular Materials (CiQUS), University of Santiago de Compostela, 15705 Santiago de Compostela, Spain
| | - Devrim Gozuacik
- Koç University Research Center for Translational Medicine (KUTTAM), Istanbul 34450, Turkey
- Department of Medical Biology, Koç University School of Medicine, Istanbul 34450, Turkey
| | - Naouale El Yamani
- Health Effects Laboratory, Department for Environmental Chemistry, NILU-Norwegian Institute for Air Research, 2027 Kjeller, Norway
| | - Tanima SenGupta
- Health Effects Laboratory, Department for Environmental Chemistry, NILU-Norwegian Institute for Air Research, 2027 Kjeller, Norway
| | - Elise Rundén-Pran
- Health Effects Laboratory, Department for Environmental Chemistry, NILU-Norwegian Institute for Air Research, 2027 Kjeller, Norway
| | - José Vivancos
- Stroke Unit, Department of Neurology, Hospital Universitario de La Princesa & Instituto de Investigación Sanitaria La Princesa, 28006 Madrid, Spain
| | - Mar Castellanos
- Neurology Service, University Hospital Complex of A Coruña, A Coruña Biomedical Research Institute, 15006 A Coruña, Spain
| | - Exuperio Díez-Tejedor
- Neurological Sciences and Cerebrovascular Research Laboratory, Department of Neurology, Neurology and Cerebrovascular Disease Group, Neuroscience Area of Hospital La Paz Institute for Health Research – IdiPAZ (La Paz University Hospital- Universidad Autónoma de Madrid), 28029 Madrid, Spain
| | - Tomás Sobrino
- NeuroAging Group (NEURAL), Clinical Neurosciences Research Laboratory (LINC), Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain
- Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, 28029 Madrid, Spain
| | - Aharon Rabinkov
- Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - David Mirelman
- Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - José Castillo
- Neuroimaging and Biotechnology Laboratory Group (NOBEL), Clinical Neurosciences Research Laboratory (LINC), Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain
| | - Francisco Campos
- Translational Stroke Laboratory Group (TREAT), Clinical Neurosciences Research Laboratory (LINC), Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain
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10
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Papadopoulos C, Arbes H, Cornu D, Chevrollier N, Blanchet S, Roginski P, Rabier C, Atia S, Lespinet O, Namy O, Lopes A. The ribosome profiling landscape of yeast reveals a high diversity in pervasive translation. Genome Biol 2024; 25:268. [PMID: 39402662 PMCID: PMC11472626 DOI: 10.1186/s13059-024-03403-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2023] [Accepted: 09/26/2024] [Indexed: 10/19/2024] Open
Abstract
BACKGROUND Pervasive translation is a widespread phenomenon that plays a critical role in the emergence of novel microproteins, but the diversity of translation patterns contributing to their generation remains unclear. Based on 54 ribosome profiling (Ribo-Seq) datasets, we investigated the yeast Ribo-Seq landscape using a representation framework that allows the comprehensive inventory and classification of the entire diversity of Ribo-Seq signals, including non-canonical ones. RESULTS We show that if coding regions occupy specific areas of the Ribo-Seq landscape, noncoding regions encompass a wide diversity of Ribo-Seq signals and, conversely, populate the entire landscape. Our results show that pervasive translation can, nevertheless, be associated with high specificity, with 1055 noncoding ORFs exhibiting canonical Ribo-Seq signals. Using mass spectrometry under standard conditions or proteasome inhibition with an in-house analysis protocol, we report 239 microproteins originating from noncoding ORFs that display canonical but also non-canonical Ribo-Seq signals. Each condition yields dozens of additional microprotein candidates with comparable translation properties, suggesting a larger population of volatile microproteins that are challenging to detect. Our findings suggest that non-canonical translation signals may harbor valuable information and underscore the significance of considering them in proteogenomic studies. Finally, we show that the translation outcome of a noncoding ORF is primarily determined by the initiating codon and the codon distribution in its two alternative frames, rather than features indicative of functionality. CONCLUSION Our results enable us to propose a topology of a species' Ribo-Seq landscape, opening the way to comparative analyses of this translation landscape under different conditions.
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Affiliation(s)
- Chris Papadopoulos
- Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, Cedex, 91198, France
- Hospital del Mar Research Institute, Barcelona, Spain
| | - Hugo Arbes
- Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, Cedex, 91198, France
| | - David Cornu
- Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, Cedex, 91198, France
| | | | - Sandra Blanchet
- Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, Cedex, 91198, France
| | - Paul Roginski
- Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, Cedex, 91198, France
| | - Camille Rabier
- Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, Cedex, 91198, France
| | - Safiya Atia
- Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, Cedex, 91198, France
| | - Olivier Lespinet
- Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, Cedex, 91198, France
| | - Olivier Namy
- Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, Cedex, 91198, France
| | - Anne Lopes
- Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, Cedex, 91198, France.
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11
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Nugent PJ, Park H, Wladyka CL, Chen KY, Bynum C, Quarterman G, Hsieh AC, Subramaniam AR. Decoding RNA Metabolism by RNA-linked CRISPR Screening in Human Cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.07.25.605204. [PMID: 39091804 PMCID: PMC11291135 DOI: 10.1101/2024.07.25.605204] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/04/2024]
Abstract
RNAs undergo a complex choreography of metabolic processes in human cells that are regulated by thousands of RNA-associated proteins. While the effects of individual RNA-associated proteins on RNA metabolism have been extensively characterized, the full complement of regulators for most RNA metabolic events remain unknown. Here we present a massively parallel RNA-linked CRISPR (ReLiC) screening approach to measure the responses of diverse RNA metabolic events to knockout of 2,092 human genes encoding all known RNA-associated proteins. ReLiC screens highlight modular interactions between gene networks regulating splicing, translation, and decay of mRNAs. When combined with biochemical fractionation of polysomes, ReLiC reveals striking pathway-specific coupling between growth fitness and mRNA translation. Perturbing different components of the translation and proteostasis machineries have distinct effects on ribosome occupancy, while perturbing mRNA transcription leaves ribosome occupancy largely intact. Isoform-selective ReLiC screens capture differential regulation of intron retention and exon skipping by SF3b complex subunits. Chemogenomic screens using ReLiC decipher translational regulators upstream of mRNA decay and uncover a role for the ribosome collision sensor GCN1 during treatment with the anti-leukemic drug homoharringtonine. Our work demonstrates ReLiC as a versatile platform for discovering and dissecting regulatory principles of human RNA metabolism.
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Affiliation(s)
- Patrick J Nugent
- Basic Sciences Division and Computational Biology Section of the Public Health Sciences Division, Fred Hutchinson Cancer Center, Seattle WA, USA
- Molecular and Cellular Biology Graduate Program, University of Washington, Seattle WA, USA
| | - Heungwon Park
- Basic Sciences Division and Computational Biology Section of the Public Health Sciences Division, Fred Hutchinson Cancer Center, Seattle WA, USA
| | - Cynthia L Wladyka
- Human Biology Division, Fred Hutchinson Cancer Center, Seattle WA, USA
| | - Katharine Y Chen
- Basic Sciences Division and Computational Biology Section of the Public Health Sciences Division, Fred Hutchinson Cancer Center, Seattle WA, USA
- Molecular and Cellular Biology Graduate Program, University of Washington, Seattle WA, USA
| | - Christine Bynum
- Basic Sciences Division and Computational Biology Section of the Public Health Sciences Division, Fred Hutchinson Cancer Center, Seattle WA, USA
- Department of Biology, Spelman College, Atlanta GA, USA
| | - Grace Quarterman
- Basic Sciences Division and Computational Biology Section of the Public Health Sciences Division, Fred Hutchinson Cancer Center, Seattle WA, USA
- Department of Biology, Spelman College, Atlanta GA, USA
| | - Andrew C Hsieh
- Human Biology Division, Fred Hutchinson Cancer Center, Seattle WA, USA
- Department of Medicine and Department of Genome Sciences, University of Washington, Seattle WA, USA
| | - Arvind Rasi Subramaniam
- Basic Sciences Division and Computational Biology Section of the Public Health Sciences Division, Fred Hutchinson Cancer Center, Seattle WA, USA
- Department of Biochemistry and Department of Genome Sciences, University of Washington, Seattle WA, USA
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12
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Mir DA, Ma Z, Horrocks J, Rogers A. Stress-Induced Eukaryotic Translational Regulatory Mechanisms. JOURNAL OF CLINICAL AND MEDICAL SCIENCES 2024; 8:1000277. [PMID: 39364184 PMCID: PMC11448810] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 10/05/2024]
Abstract
The eukaryotic protein synthesis process entails intricate stages governed by diverse mechanisms to tightly regulate translation. Translational regulation during stress is pivotal for maintaining cellular homeostasis, ensuring the accurate expression of essential proteins is important for survival. This selective translational control mechanism is integral to cellular adaptation and resilience under adverse conditions. This review manuscript explores various mechanisms involved in selective translational regulation, focusing on mRNA-specific and global regulatory processes. Key aspects of translational control include translation initiation, which is often a rate-limiting step, and involves the formation of the eIF4F complex and recruitment of mRNA to ribosomes. Regulation of translation initiation factors, such as eIF4E, eIF4E2, and eIF2, through phosphorylation and interactions with binding proteins, modulates translation efficiency under stress conditions. This review also highlights the control of translation initiation through factors like the eIF4F complex and the ternary complex and also underscores the importance of eIF2α phosphorylation in stress granule formation and cellular stress responses. Additionally, the impact of amino acid deprivation, mTOR signaling, and ribosome biogenesis on translation regulation and cellular adaptation to stress is also discussed. Understanding the intricate mechanisms of translational regulation during stress provides insights into cellular adaptation mechanisms and potential therapeutic targets for various diseases, offering valuable avenues for addressing conditions associated with dysregulated protein synthesis.
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Affiliation(s)
- Dilawar Ahmad Mir
- Kathryn W. Davis Center for Regenerative Biology and Aging, Mount Desert Island Biological Laboratory, Maine, United States of America
| | - Zhengxin Ma
- Kathryn W. Davis Center for Regenerative Biology and Aging, Mount Desert Island Biological Laboratory, Maine, United States of America
| | - Jordan Horrocks
- Kathryn W. Davis Center for Regenerative Biology and Aging, Mount Desert Island Biological Laboratory, Maine, United States of America
| | - Aric Rogers
- Kathryn W. Davis Center for Regenerative Biology and Aging, Mount Desert Island Biological Laboratory, Maine, United States of America
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13
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Yadav P, Tamilselvan R, Mani H, Singh KK. MicroRNA-mediated regulation of nonsense-mediated mRNA decay factors: Insights into microRNA prediction tools and profiling techniques. BIOCHIMICA ET BIOPHYSICA ACTA. GENE REGULATORY MECHANISMS 2024; 1867:195022. [PMID: 38437914 DOI: 10.1016/j.bbagrm.2024.195022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/20/2023] [Revised: 02/28/2024] [Accepted: 03/01/2024] [Indexed: 03/06/2024]
Abstract
Nonsense-mediated mRNA decay (NMD) stands out as a prominent RNA surveillance mechanism within eukaryotes, meticulously overseeing both RNA abundance and integrity by eliminating aberrant transcripts. These defective transcripts are discerned through the concerted efforts of translating ribosomes, eukaryotic release factors (eRFs), and trans-acting NMD factors, with Up-Frameshift 3 (UPF3) serving as a noteworthy component. Remarkably, in humans, UPF3 exists in two paralogous forms, UPF3A (UPF3) and UPF3B (UPF3X). Beyond its role in quality control, UPF3 wields significant influence over critical cellular processes, including neural development, synaptic plasticity, and axon guidance. However, the precise regulatory mechanisms governing UPF3 remain elusive. MicroRNAs (miRNAs) emerge as pivotal post-transcriptional gene regulators, exerting substantial impact on diverse pathological and physiological pathways. This comprehensive review encapsulates our current understanding of the intricate regulatory nexus between NMD and miRNAs, with particular emphasis on the essential role played by UPF3B in neurodevelopment. Additionally, we bring out the significance of the 3'-untranslated region (3'-UTR) as the molecular bridge connecting NMD and miRNA-mediated gene regulation. Furthermore, we provide an in-depth exploration of diverse computational tools tailored for the prediction of potential miRNA targets. To complement these computational approaches, we delineate experimental techniques designed to validate predicted miRNA-mRNA interactions, empowering readers with the knowledge necessary to select the most appropriate methodology for their specific research objectives.
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Affiliation(s)
- Priyanka Yadav
- Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India
| | - Raja Tamilselvan
- Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India
| | - Harita Mani
- Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India
| | - Kusum Kumari Singh
- Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India.
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14
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Dasgupta A, Prensner JR. Upstream open reading frames: new players in the landscape of cancer gene regulation. NAR Cancer 2024; 6:zcae023. [PMID: 38774471 PMCID: PMC11106035 DOI: 10.1093/narcan/zcae023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2023] [Revised: 04/29/2024] [Accepted: 05/07/2024] [Indexed: 05/24/2024] Open
Abstract
The translation of RNA by ribosomes represents a central biological process and one of the most dysregulated processes in cancer. While translation is traditionally thought to occur exclusively in the protein-coding regions of messenger RNAs (mRNAs), recent transcriptome-wide approaches have shown abundant ribosome activity across diverse stretches of RNA transcripts. The most common type of this kind of ribosome activity occurs in gene leader sequences, also known as 5' untranslated regions (UTRs) of the mRNA, that precede the main coding sequence. Translation of these upstream open reading frames (uORFs) is now known to occur in upwards of 25% of all protein-coding genes. With diverse functions from RNA regulation to microprotein generation, uORFs are rapidly igniting a new arena of cancer biology, where they are linked to cancer genetics, cancer signaling, and tumor-immune interactions. This review focuses on the contributions of uORFs and their associated 5'UTR sequences to cancer biology.
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Affiliation(s)
- Anwesha Dasgupta
- Chad Carr Pediatric Brain Tumor Center, University of Michigan Medical School, Ann Arbor, MI 48109, USA
- Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
- Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - John R Prensner
- Chad Carr Pediatric Brain Tumor Center, University of Michigan Medical School, Ann Arbor, MI 48109, USA
- Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
- Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA
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15
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Andjus S, Szachnowski U, Vogt N, Gioftsidi S, Hatin I, Cornu D, Papadopoulos C, Lopes A, Namy O, Wery M, Morillon A. Pervasive translation of Xrn1-sensitive unstable long noncoding RNAs in yeast. RNA (NEW YORK, N.Y.) 2024; 30:662-679. [PMID: 38443115 PMCID: PMC11098462 DOI: 10.1261/rna.079903.123] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/29/2023] [Accepted: 02/15/2024] [Indexed: 03/07/2024]
Abstract
Despite being predicted to lack coding potential, cytoplasmic long noncoding (lnc)RNAs can associate with ribosomes. However, the landscape and biological relevance of lncRNA translation remain poorly studied. In yeast, cytoplasmic Xrn1-sensitive unstable transcripts (XUTs) are targeted by nonsense-mediated mRNA decay (NMD), suggesting a translation-dependent degradation process. Here, we report that XUTs are pervasively translated, which impacts their decay. We show that XUTs globally accumulate upon translation elongation inhibition, but not when initial ribosome loading is impaired. Ribo-seq confirmed ribosomes binding to XUTs and identified ribosome-associated 5'-proximal small ORFs. Mechanistically, the NMD-sensitivity of XUTs mainly depends on the 3'-untranslated region length. Finally, we show that the peptide resulting from the translation of an NMD-sensitive XUT reporter exists in NMD-competent cells. Our work highlights the role of translation in the posttranscriptional metabolism of XUTs. We propose that XUT-derived peptides could be exposed to natural selection, while NMD restricts XUT levels.
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Affiliation(s)
- Sara Andjus
- ncRNA, Epigenetic and Genome Fluidity, Institut Curie, PSL University, Sorbonne Université, CNRS UMR3244, F-75248 Paris Cedex 05, France
| | - Ugo Szachnowski
- ncRNA, Epigenetic and Genome Fluidity, Institut Curie, Sorbonne Université, CNRS UMR3244, F-75248 Paris Cedex 05, France
| | - Nicolas Vogt
- ncRNA, Epigenetic and Genome Fluidity, Institut Curie, Sorbonne Université, CNRS UMR3244, F-75248 Paris Cedex 05, France
| | - Stamatia Gioftsidi
- ncRNA, Epigenetic and Genome Fluidity, Institut Curie, Sorbonne Université, CNRS UMR3244, F-75248 Paris Cedex 05, France
| | - Isabelle Hatin
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France
| | - David Cornu
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France
| | - Chris Papadopoulos
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France
| | - Anne Lopes
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France
| | - Olivier Namy
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France
| | - Maxime Wery
- ncRNA, Epigenetic and Genome Fluidity, Institut Curie, Sorbonne Université, CNRS UMR3244, F-75248 Paris Cedex 05, France
| | - Antonin Morillon
- ncRNA, Epigenetic and Genome Fluidity, Institut Curie, Sorbonne Université, CNRS UMR3244, F-75248 Paris Cedex 05, France
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16
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Ma Z, Sharma R, Rogers AN. Physiological Consequences of Nonsense-Mediated Decay and Its Role in Adaptive Responses. Biomedicines 2024; 12:1110. [PMID: 38791071 PMCID: PMC11117581 DOI: 10.3390/biomedicines12051110] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2024] [Revised: 04/30/2024] [Accepted: 05/13/2024] [Indexed: 05/26/2024] Open
Abstract
The evolutionarily conserved nonsense-mediated mRNA decay (NMD) pathway is a quality control mechanism that degrades aberrant mRNA containing one or more premature termination codons (PTCs). Recent discoveries indicate that NMD also differentially regulates mRNA from wild-type protein-coding genes despite lacking PTCs. Together with studies showing that NMD is involved in development and adaptive responses that influence health and longevity, these findings point to an expanded role of NMD that adds a new layer of complexity in the post-transcriptional regulation of gene expression. However, the extent of its control, whether different types of NMD play different roles, and the resulting physiological outcomes remain unclear and need further elucidation. Here, we review different branches of NMD and what is known of the physiological outcomes associated with this type of regulation. We identify significant gaps in the understanding of this process and the utility of genetic tools in accelerating progress in this area.
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Affiliation(s)
- Zhengxin Ma
- MDI Biological Laboratory, Bar Harbor, ME 04609, USA
| | - Ratna Sharma
- Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA;
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17
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Mir DA, Ma Z, Horrocks J, Rogers AN. Stress-induced Eukaryotic Translational Regulatory Mechanisms. ARXIV 2024:arXiv:2405.01664v1. [PMID: 38745702 PMCID: PMC11092689] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 05/16/2024]
Abstract
The eukaryotic protein synthesis process entails intricate stages governed by diverse mechanisms to tightly regulate translation. Translational regulation during stress is pivotal for maintaining cellular homeostasis, ensuring the accurate expression of essential proteins crucial for survival. This selective translational control mechanism is integral to cellular adaptation and resilience under adverse conditions. This review manuscript explores various mechanisms involved in selective translational regulation, focusing on mRNA-specific and global regulatory processes. Key aspects of translational control include translation initiation, which is often a rate-limiting step, and involves the formation of the eIF4F complex and recruitment of mRNA to ribosomes. Regulation of translation initiation factors, such as eIF4E, eIF4E2, and eIF2, through phosphorylation and interactions with binding proteins, modulates translation efficiency under stress conditions. This review also highlights the control of translation initiation through factors like the eIF4F complex and the ternary complex and also underscores the importance of eIF2α phosphorylation in stress granule formation and cellular stress responses. Additionally, the impact of amino acid deprivation, mTOR signaling, and ribosome biogenesis on translation regulation and cellular adaptation to stress is also discussed. Understanding the intricate mechanisms of translational regulation during stress provides insights into cellular adaptation mechanisms and potential therapeutic targets for various diseases, offering valuable avenues for addressing conditions associated with dysregulated protein synthesis.
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Affiliation(s)
- Dilawar Ahmad Mir
- Kathryn W. Davis Center for Regenerative Biology and Aging, Mount Desert Island Biological Laboratory, Bar Harbor, ME
| | - Zhengxin Ma
- Kathryn W. Davis Center for Regenerative Biology and Aging, Mount Desert Island Biological Laboratory, Bar Harbor, ME
| | - Jordan Horrocks
- Kathryn W. Davis Center for Regenerative Biology and Aging, Mount Desert Island Biological Laboratory, Bar Harbor, ME
| | - Aric N Rogers
- Kathryn W. Davis Center for Regenerative Biology and Aging, Mount Desert Island Biological Laboratory, Bar Harbor, ME
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18
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Kolakada D, Campbell AE, Galvis LB, Li Z, Lore M, Jagannathan S. A system of reporters for comparative investigation of EJC-independent and EJC-enhanced nonsense-mediated mRNA decay. Nucleic Acids Res 2024; 52:e34. [PMID: 38375914 PMCID: PMC11014337 DOI: 10.1093/nar/gkae121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2023] [Revised: 01/05/2024] [Accepted: 02/07/2024] [Indexed: 02/21/2024] Open
Abstract
Nonsense-mediated mRNA decay (NMD) is a network of pathways that degrades transcripts that undergo premature translation termination. In mammals, NMD can be divided into the exon junction complex (EJC)-enhanced and EJC-independent branches. Fluorescence- and luminescence-based reporters have long been effective tools to investigate NMD, yet existing reporters largely focus on the EJC-enhanced pathway. Here, we present a system of reporters for comparative studies of EJC-independent and EJC-enhanced NMD. This system also enables the study of NMD-associated outcomes such as premature termination codon (PTC) readthrough and truncated protein degradation. These reporters are compatible with fluorescence or luminescence-based readouts via transient transfection or stable integration. Using this reporter system, we show that EJC-enhanced NMD RNA levels are reduced by 2- or 9-fold and protein levels are reduced by 7- or 12-fold compared to EJC-independent NMD, depending on the reporter gene used. Additionally, the extent of readthrough induced by G418 and an NMD inhibitor (SMG1i), alone and in combination, varies across NMD substrates. When combined, G418 and SMG1i increase readthrough product levels in an additive manner for EJC-independent reporters, while EJC-enhanced reporters show a synergistic effect. We present these reporters as a valuable toolkit to deepen our understanding of NMD and its associated mechanisms.
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Affiliation(s)
- Divya Kolakada
- Molecular Biology Graduate Program, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
- Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Amy E Campbell
- Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Laura Baquero Galvis
- Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Zhongyou Li
- Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Mlana Lore
- Molecular Biology Graduate Program, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
- Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Sujatha Jagannathan
- Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
- RNA Bioscience Initiative, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
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19
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Smirnova AM, Hronova V, Mohammad MP, Herrmannova A, Gunisova S, Petrackova D, Halada P, Coufal S, Swirski M, Rendelman J, Jendruchova K, Hatzoglou M, Beznoskova P, Vogel C, Valasek LS. Stem-loop induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2023.07.12.548609. [PMID: 37502919 PMCID: PMC10369994 DOI: 10.1101/2023.07.12.548609] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/29/2023]
Abstract
ATF4 is a master transcriptional regulator of the integrated stress response leading cells towards adaptation or death. ATF4's induction under stress was thought to be mostly due to delayed translation reinitiation, where the reinitiation-permissive uORF1 plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations, but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrate that the canonical ATF4 translation start site is substantially leaky-scanned. Thus, ATF4's translational control is more complex than originally described underpinning its key role in diverse biological processes.
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20
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Filippopoulou C, Thomé CC, Perdikari S, Ntini E, Simos G, Bohnsack KE, Chachami G. Hypoxia-driven deSUMOylation of EXOSC10 promotes adaptive changes in the transcriptome profile. Cell Mol Life Sci 2024; 81:58. [PMID: 38279024 PMCID: PMC10817850 DOI: 10.1007/s00018-023-05035-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2023] [Revised: 10/12/2023] [Accepted: 11/06/2023] [Indexed: 01/28/2024]
Abstract
Reduced oxygen availability (hypoxia) triggers adaptive cellular responses via hypoxia-inducible factor (HIF)-dependent transcriptional activation. Adaptation to hypoxia also involves transcription-independent processes like post-translational modifications; however, these mechanisms are poorly characterized. Investigating the involvement of protein SUMOylation in response to hypoxia, we discovered that hypoxia strongly decreases the SUMOylation of Exosome subunit 10 (EXOSC10), the catalytic subunit of the RNA exosome, in an HIF-independent manner. EXOSC10 is a multifunctional exoribonuclease enriched in the nucleolus that mediates the processing and degradation of various RNA species. We demonstrate that the ubiquitin-specific protease 36 (USP36) SUMOylates EXOSC10 and we reveal SUMO1/sentrin-specific peptidase 3 (SENP3) as the enzyme-mediating deSUMOylation of EXOSC10. Under hypoxia, EXOSC10 dissociates from USP36 and translocates from the nucleolus to the nucleoplasm concomitant with its deSUMOylation. Loss of EXOSC10 SUMOylation does not detectably affect rRNA maturation but affects the mRNA transcriptome by modulating the expression levels of hypoxia-related genes. Our data suggest that dynamic modulation of EXOSC10 SUMOylation and localization under hypoxia regulates the RNA degradation machinery to facilitate cellular adaptation to low oxygen conditions.
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Affiliation(s)
- Chrysa Filippopoulou
- Laboratory of Biochemistry, Faculty of Medicine, University of Thessaly, Biopolis, 41500, Larissa, Greece
| | - Chairini C Thomé
- Department of Molecular Biology, University Medical Center Göttingen, 37073, Göttingen, Germany
| | - Sofia Perdikari
- Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas (FORTH), 70013, Heraklion, Greece
| | - Evgenia Ntini
- Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas (FORTH), 70013, Heraklion, Greece
| | - George Simos
- Laboratory of Biochemistry, Faculty of Medicine, University of Thessaly, Biopolis, 41500, Larissa, Greece
- Gerald Bronfman Department of Oncology, Faculty of Medicine, McGill University, Montreal, Canada
| | - Katherine E Bohnsack
- Department of Molecular Biology, University Medical Center Göttingen, 37073, Göttingen, Germany
| | - Georgia Chachami
- Laboratory of Biochemistry, Faculty of Medicine, University of Thessaly, Biopolis, 41500, Larissa, Greece.
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21
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Zavileyskiy LG, Pervouchine DD. Post-transcriptional Regulation of Gene Expression via Unproductive Splicing. Acta Naturae 2024; 16:4-13. [PMID: 38698955 PMCID: PMC11062102 DOI: 10.32607/actanaturae.27337] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2023] [Accepted: 03/01/2024] [Indexed: 05/05/2024] Open
Abstract
Unproductive splicing is a mechanism of post-transcriptional gene expression control in which premature stop codons are inserted into protein-coding transcripts as a result of regulated alternative splicing, leading to their degradation via the nonsense-mediated decay pathway. This mechanism is especially characteristic of RNA-binding proteins, which regulate each other's expression levels and those of other genes in multiple auto- and cross-regulatory loops. Deregulation of unproductive splicing is a cause of serious human diseases, including cancers, and is increasingly being considered as a prominent therapeutic target. This review discusses the types of unproductive splicing events, the mechanisms of auto- and cross-regulation, nonsense-mediated decay escape, and problems in identifying unproductive splice isoforms. It also provides examples of deregulation of unproductive splicing in human diseases and discusses therapeutic strategies for its correction using antisense oligonucleotides and small molecules.
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Affiliation(s)
- L. G. Zavileyskiy
- Lomonosov Moscow State University, Moscow, 119192 Russian Federation
- Skolkovo Institute of Science and Technology, Moscow, 121205 Russian Federation
| | - D. D. Pervouchine
- Skolkovo Institute of Science and Technology, Moscow, 121205 Russian Federation
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22
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Ma Z, Horrocks J, Mir DA, Cox M, Ruzga M, Rollins J, Rogers AN. The integrated stress response protects against ER stress but is not required for altered translation and lifespan from dietary restriction in Caenorhabditis elegans. Front Cell Dev Biol 2023; 11:1263344. [PMID: 38161330 PMCID: PMC10755965 DOI: 10.3389/fcell.2023.1263344] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2023] [Accepted: 11/24/2023] [Indexed: 01/03/2024] Open
Abstract
The highly conserved integrated stress response (ISR) reduces and redirects mRNA translation in response to certain forms of stress and nutrient limitation. It is activated when kinases phosphorylate a key residue in the alpha subunit of eukaryotic translation initiation factor 2 (eIF2). General Control Nonderepressible-2 (GCN2) is activated to phosphorylate eIF2α by the presence of uncharged tRNA associated with nutrient scarcity, while protein kinase R-like ER kinase-1 (PERK) is activated during the ER unfolded protein response (UPRER). Here, we investigated the role of the ISR during nutrient limitation and ER stress with respect to changes in protein synthesis, translationally driven mRNA turnover, and survival in Caenorhabditis elegans. We found that, while GCN2 phosphorylates eIF2α when nutrients are restricted, the ability to phosphorylate eIF2α is not required for changes in translation, nonsense-mediated decay, or lifespan associated with dietary restriction (DR). Interestingly, loss of both GCN2 and PERK abolishes increased lifespan associated with dietary restriction, indicating the possibility of other substrates for these kinases. The ISR was not dispensable under ER stress conditions, as demonstrated by the requirement for PERK and eIF2α phosphorylation for decreased translation and wild type-like survival. Taken together, results indicate that the ISR is critical for ER stress and that other translation regulatory mechanisms are sufficient for increased lifespan under dietary restriction.
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Affiliation(s)
| | | | | | | | | | | | - Aric N. Rogers
- MDI Biological Laboratory, Bar Harbor, ME, United States
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23
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Burkart V, Kowalski K, Disch A, Hilfiker-Kleiner D, Lal S, Dos Remedios C, Perrot A, Zeug A, Ponimaskin E, Kosanke M, Dittrich-Breiholz O, Kraft T, Montag J. Nonsense mediated decay factor UPF3B is associated with cMyBP-C haploinsufficiency in hypertrophic cardiomyopathy patients. J Mol Cell Cardiol 2023; 185:26-37. [PMID: 37797718 DOI: 10.1016/j.yjmcc.2023.09.008] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/15/2023] [Revised: 09/08/2023] [Accepted: 09/28/2023] [Indexed: 10/07/2023]
Abstract
Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiac disease. Up to 40% of cases are associated with heterozygous mutations in myosin binding protein C (cMyBP-C, MYBPC3). Most of these mutations lead to premature termination codons (PTC) and patients show reduction of functional cMyBP-C. This so-called haploinsufficiency most likely contributes to disease development. We analyzed mechanisms underlying haploinsufficiency using cardiac tissue from HCM-patients with truncation mutations in MYBPC3 (MYBPC3trunc). We compared transcriptional activity, mRNA and protein expression to donor controls. To differentiate between HCM-specific and general hypertrophy-induced mechanisms we used patients with left ventricular hypertrophy due to aortic stenosis (AS) as an additional control. We show that cMyBP-C haploinsufficiency starts at the mRNA level, despite hypertrophy-induced increased transcriptional activity. Gene set enrichment analysis (GSEA) of RNA-sequencing data revealed an increased expression of NMD-components. Among them, Up-frameshift protein UPF3B, a regulator of NMD was upregulated in MYBPC3trunc patients and not in AS-patients. Strikingly, we show that in sarcomeres UPF3B but not UPF1 and UPF2 are localized to the Z-discs, the presumed location of sarcomeric protein translation. Our data suggest that cMyBP-C haploinsufficiency in HCM-patients is established by UPF3B-dependent NMD during the initial translation round at the Z-disc.
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Affiliation(s)
- Valentin Burkart
- Institute for Molecular and Cell Physiology, Hannover Medical School, Hannover, Germany.
| | - Kathrin Kowalski
- Institute for Molecular and Cell Physiology, Hannover Medical School, Hannover, Germany
| | - Alina Disch
- Institute for Molecular and Cell Physiology, Hannover Medical School, Hannover, Germany
| | | | - Sean Lal
- School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Sydney, Australia
| | - Cristobal Dos Remedios
- Mechanosensory Biophysics Laboratory, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia
| | - Andreas Perrot
- Charité - Universitätsmedizin Berlin, Experimental & Clinical Research Center, Berlin, Germany
| | - Andre Zeug
- Institute of Neurophysiology, Hannover Medical School, Hannover, Germany
| | - Evgeni Ponimaskin
- Institute of Neurophysiology, Hannover Medical School, Hannover, Germany
| | - Maike Kosanke
- Research Core Unit Genomics, Hannover Medical School, Hannover, Germany
| | | | - Theresia Kraft
- Institute for Molecular and Cell Physiology, Hannover Medical School, Hannover, Germany
| | - Judith Montag
- Institute for Molecular and Cell Physiology, Hannover Medical School, Hannover, Germany
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24
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Kolakada D, Campbell AE, Baquero Galvis L, Li Z, Lore M, Jagannathan S. A system of reporters for comparative investigation of EJC-independent and EJC-enhanced nonsense-mediated mRNA decay. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.11.14.567061. [PMID: 38014198 PMCID: PMC10680754 DOI: 10.1101/2023.11.14.567061] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/29/2023]
Abstract
Nonsense-mediated mRNA decay (NMD) is a network of pathways that degrades transcripts that undergo premature translation termination. In mammals, NMD can be divided into the exon junction complex (EJC)-enhanced and EJC-independent branches. Fluorescence- and luminescence-based reporters have long been effective tools to investigate NMD, yet existing reporters largely focus on the EJC-enhanced pathway. Here, we present a system of reporters for comparative studies of EJC-independent and EJC-enhanced NMD. This system also enables the study of NMD-associated outcomes such as premature termination codon (PTC) readthrough and truncated protein degradation. These reporters are compatible with fluorescence or luminescence-based readouts via transient transfection or stable integration. Using this reporter system, we show that EJC-enhanced NMD RNA levels are reduced by 2- or 9-fold and protein levels are reduced by 7- or 12-fold compared to EJC-independent NMD, depending on the reporter gene used. Additionally, the extent of readthrough induced by G418 and SMG1i, alone and in combination, varies across NMD substrates. When combined, G418 and SMG1i increase readthrough product levels in an additive manner for EJC-independent reporters, while EJC-enhanced reporters show a synergistic effect. We present these reporters as a valuable toolkit to deepen our understanding of NMD and its associated mechanisms.
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Affiliation(s)
- Divya Kolakada
- Molecular Biology Graduate Program, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
- Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Amy E. Campbell
- Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Laura Baquero Galvis
- Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Zhongyou Li
- Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Mlana Lore
- Molecular Biology Graduate Program, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
- Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Sujatha Jagannathan
- Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
- RNA Bioscience Initiative, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
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25
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Vujovic F, Shepherd CE, Witting PK, Hunter N, Farahani RM. Redox-Mediated Rewiring of Signalling Pathways: The Role of a Cellular Clock in Brain Health and Disease. Antioxidants (Basel) 2023; 12:1873. [PMID: 37891951 PMCID: PMC10604469 DOI: 10.3390/antiox12101873] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2023] [Revised: 10/14/2023] [Accepted: 10/15/2023] [Indexed: 10/29/2023] Open
Abstract
Metazoan signalling pathways can be rewired to dampen or amplify the rate of events, such as those that occur in development and aging. Given that a linear network topology restricts the capacity to rewire signalling pathways, such scalability of the pace of biological events suggests the existence of programmable non-linear elements in the underlying signalling pathways. Here, we review the network topology of key signalling pathways with a focus on redox-sensitive proteins, including PTEN and Ras GTPase, that reshape the connectivity profile of signalling pathways in response to an altered redox state. While this network-level impact of redox is achieved by the modulation of individual redox-sensitive proteins, it is the population by these proteins of critical nodes in a network topology of signal transduction pathways that amplifies the impact of redox-mediated reprogramming. We propose that redox-mediated rewiring is essential to regulate the rate of transmission of biological signals, giving rise to a programmable cellular clock that orchestrates the pace of biological phenomena such as development and aging. We further review the evidence that an aberrant redox-mediated modulation of output of the cellular clock contributes to the emergence of pathological conditions affecting the human brain.
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Affiliation(s)
- Filip Vujovic
- IDR/Westmead Institute for Medical Research, Sydney, NSW 2145, Australia; (F.V.); (N.H.)
- School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia
| | | | - Paul K. Witting
- Redox Biology Group, Charles Perkins Centre, Faculty of Medicine and Health, School of Medical Sciences, The University of Sydney, Sydney, NSW 2006, Australia;
| | - Neil Hunter
- IDR/Westmead Institute for Medical Research, Sydney, NSW 2145, Australia; (F.V.); (N.H.)
| | - Ramin M. Farahani
- IDR/Westmead Institute for Medical Research, Sydney, NSW 2145, Australia; (F.V.); (N.H.)
- School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia
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26
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Monaghan L, Longman D, Cáceres JF. Translation-coupled mRNA quality control mechanisms. EMBO J 2023; 42:e114378. [PMID: 37605642 PMCID: PMC10548175 DOI: 10.15252/embj.2023114378] [Citation(s) in RCA: 31] [Impact Index Per Article: 15.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2023] [Revised: 07/24/2023] [Accepted: 08/01/2023] [Indexed: 08/23/2023] Open
Abstract
mRNA surveillance pathways are essential for accurate gene expression and to maintain translation homeostasis, ensuring the production of fully functional proteins. Future insights into mRNA quality control pathways will enable us to understand how cellular mRNA levels are controlled, how defective or unwanted mRNAs can be eliminated, and how dysregulation of these can contribute to human disease. Here we review translation-coupled mRNA quality control mechanisms, including the non-stop and no-go mRNA decay pathways, describing their mechanisms, shared trans-acting factors, and differences. We also describe advances in our understanding of the nonsense-mediated mRNA decay (NMD) pathway, highlighting recent mechanistic findings, the discovery of novel factors, as well as the role of NMD in cellular physiology and its impact on human disease.
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Affiliation(s)
- Laura Monaghan
- MRC Human Genetics Unit, Institute of Genetics and CancerUniversity of EdinburghEdinburghUK
| | - Dasa Longman
- MRC Human Genetics Unit, Institute of Genetics and CancerUniversity of EdinburghEdinburghUK
| | - Javier F Cáceres
- MRC Human Genetics Unit, Institute of Genetics and CancerUniversity of EdinburghEdinburghUK
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27
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Steiner AJ, Zheng Y, Tang Y. Characterization of a rhabdomyosarcoma reveals a critical role for SMG7 in cancer cell viability and tumor growth. Sci Rep 2023; 13:10152. [PMID: 37349371 PMCID: PMC10287741 DOI: 10.1038/s41598-023-36568-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2023] [Accepted: 06/06/2023] [Indexed: 06/24/2023] Open
Abstract
Soft-tissue sarcomas (STSs) are a rare and diverse group of mesenchymal cancers plagued with aggression, poor response to systemic therapy, and high rates of recurrence. Although STSs generally have low mutational burdens, the most commonly mutated genes are tumor suppressors, which frequently acquire mutations inducing nonsense-mediated mRNA decay (NMD). This suggests that STS cells may exploit NMD to suppress these anti-cancer genes. To examine the role that the NMD factor SMG7 plays in STS, we developed an inducible knockout mouse model in the Trp53-/- background. Here, we isolated a subcutaneous STS and identified it as a rhabdomyosarcoma (RMS). We report that knockout of SMG7 significantly inhibited NMD in our RMS cells, which led to the induction of NMD targets GADD45b and the tumor suppressor GAS5. The loss of NMD and upregulation of these anti-cancer genes were concomitant with the loss of RMS cell viability and inhibited tumor growth. Importantly, SMG7 was dispensable for homeostasis in our mouse embryonic fibroblasts and adult mice. Overall, our data show that the loss of SMG7 induces a strong anti-cancer effect both in vitro and in vivo. We present here the first evidence that disrupting SMG7 function may be tolerable and provide a therapeutic benefit for STS treatment.
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Affiliation(s)
- Alexander J Steiner
- Department of Regenerative and Cancer Cell Biology, Albany Medical College, 47 New Scotland Avenue, Albany, NY, 12208, USA
| | - Yang Zheng
- Department of Regenerative and Cancer Cell Biology, Albany Medical College, 47 New Scotland Avenue, Albany, NY, 12208, USA
| | - Yi Tang
- Department of Regenerative and Cancer Cell Biology, Albany Medical College, 47 New Scotland Avenue, Albany, NY, 12208, USA.
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28
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Russell PJ, Slivka JA, Boyle EP, Burghes AHM, Kearse MG. Translation reinitiation after uORFs does not fully protect mRNAs from nonsense-mediated decay. RNA (NEW YORK, N.Y.) 2023; 29:735-744. [PMID: 36878710 PMCID: PMC10187673 DOI: 10.1261/rna.079525.122] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/03/2022] [Accepted: 02/14/2023] [Indexed: 05/18/2023]
Abstract
It is estimated that nearly 50% of mammalian transcripts contain at least one upstream open reading frame (uORF), which are typically one to two orders of magnitude smaller than the downstream main ORF. Most uORFs are thought to be inhibitory as they sequester the scanning ribosome, but in some cases allow for translation reinitiation. However, termination in the 5' UTR at the end of uORFs resembles premature termination that is normally sensed by the nonsense-mediated mRNA decay (NMD) pathway. Translation reinitiation has been proposed as a method for mRNAs to prevent NMD. Here, we test how uORF length influences translation reinitiation and mRNA stability in HeLa cells. Using custom 5' UTRs and uORF sequences, we show that reinitiation can occur on heterologous mRNA sequences, favors small uORFs, and is supported when initiation occurs with more initiation factors. After determining reporter mRNA half-lives in HeLa cells and mining available mRNA half-life data sets for cumulative predicted uORF length, we conclude that translation reinitiation after uORFs is not a robust method for mRNAs to prevent NMD. Together, these data suggest that the decision of whether NMD ensues after translating uORFs occurs before reinitiation in mammalian cells.
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Affiliation(s)
- Paul J Russell
- Cellular, Molecular, and Biochemical Sciences Program, The Ohio State University, Columbus, Ohio 43210, USA
- The Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA
- Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, Ohio 43210, USA
- Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA
| | - Jacob A Slivka
- Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, Ohio 43210, USA
- Department of Computer Science and Engineering, The Ohio State University, Columbus, Ohio 43210, USA
| | - Elaina P Boyle
- Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, Ohio 43210, USA
- Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA
| | - Arthur H M Burghes
- Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, Ohio 43210, USA
| | - Michael G Kearse
- Cellular, Molecular, and Biochemical Sciences Program, The Ohio State University, Columbus, Ohio 43210, USA
- The Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA
- Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, Ohio 43210, USA
- Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA
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29
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Capobianco E, McGaughey V, Seraphin G, Heckel J, Rieger S, Lisse TS. Vitamin D inhibits osteosarcoma by reprogramming nonsense-mediated RNA decay and SNAI2-mediated epithelial-to-mesenchymal transition. Front Oncol 2023; 13:1188641. [PMID: 37228489 PMCID: PMC10203545 DOI: 10.3389/fonc.2023.1188641] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2023] [Accepted: 04/20/2023] [Indexed: 05/27/2023] Open
Abstract
Osteosarcomas are immune-resistant and metastatic as a result of elevated nonsense-mediated RNA decay (NMD), reactive oxygen species (ROS), and epithelial-to-mesenchymal transition (EMT). Although vitamin D has anti-cancer effects, its effectiveness and mechanism of action against osteosarcomas are poorly understood. In this study, we assessed the impact of vitamin D and its receptor (VDR) on NMD-ROS-EMT signaling in in vitro and in vivo osteosarcoma animal models. Initiation of VDR signaling facilitated the enrichment of EMT pathway genes, after which 1,25(OH)2D, the active vitamin D derivative, inhibited the EMT pathway in osteosarcoma subtypes. The ligand-bound VDR directly downregulated the EMT inducer SNAI2, differentiating highly metastatic from low metastatic subtypes and 1,25(OH)2D sensitivity. Moreover, epigenome-wide motif and putative target gene analysis revealed the VDR's integration with NMD tumorigenic and immunogenic pathways. In an autoregulatory manner, 1,25(OH)2D inhibited NMD machinery genes and upregulated NMD target genes implicated in anti-oncogenic activity, immunorecognition, and cell-to-cell adhesion. Dicer substrate siRNA knockdown of SNAI2 revealed superoxide dismutase 2 (SOD2)-mediated antioxidative responses and 1,25(OH)2D sensitization via non-canonical SOD2 nuclear-to-mitochondrial translocalization leading to overall ROS suppression. In a mouse xenograft metastasis model, the therapeutically relevant vitamin D derivative calcipotriol inhibited osteosarcoma metastasis and tumor growth shown for the first time. Our results uncover novel osteosarcoma-inhibiting mechanisms for vitamin D and calcipotriol that may be translated to human patients.
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Affiliation(s)
| | - Vanessa McGaughey
- Department of Biology, University of Miami, Coral Gables, FL, United States
| | - Gerbenn Seraphin
- Department of Biology, University of Miami, Coral Gables, FL, United States
| | - John Heckel
- Department of Biology, University of Miami, Coral Gables, FL, United States
| | - Sandra Rieger
- Department of Biology, University of Miami, Coral Gables, FL, United States
- Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, FL, United States
| | - Thomas S. Lisse
- Department of Biology, University of Miami, Coral Gables, FL, United States
- Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, FL, United States
- iCURA DX, Malvern, PA, United States
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30
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Capobianco E, McGaughey V, Seraphin G, Heckel J, Rieger S, Lisse TS. Vitamin D inhibits osteosarcoma by reprogramming nonsense-mediated RNA decay and SNAI2-mediated epithelial-to-mesenchymal transition. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.01.04.522778. [PMID: 36711643 PMCID: PMC9882006 DOI: 10.1101/2023.01.04.522778] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Osteosarcomas are immune-resistant and metastatic as a result of elevated nonsense-mediated RNA decay (NMD), reactive oxygen species (ROS), and epithelial-to-mesenchymal transition (EMT). Although vitamin D has anti-cancer effects, its effectiveness and mechanism of action against osteosarcomas are poorly understood. In this study, we assessed the impact of vitamin D and its receptor (VDR) on the NMD-ROS-EMT signaling axis in in vitro and in vivo osteosarcoma animal models. Initiation of VDR signaling facilitated the enrichment of EMT pathway genes, after which 1,25(OH) 2 D, the active vitamin D derivative, inhibited the EMT pathway in osteosarcoma subtypes. The ligand-bound VDR directly downregulated the EMT inducer SNAI2 , differentiating highly metastatic from low metastatic subtypes and 1,25(OH) 2 D sensitivity. Moreover, epigenome-wide motif and putative target gene analysis revealed the VDR’s integration with NMD tumorigenic and immunogenic pathways. In an autoregulatory manner, 1,25(OH) 2 D inhibited NMD machinery genes and upregulated NMD target genes implicated in anti-oncogenic activity, immunorecognition, and cell-to-cell adhesion. Dicer substrate siRNA knockdown of SNAI2 revealed superoxide dismutase 2 (SOD2)-mediated antioxidative responses and 1,25(OH) 2 D sensitization via non-canonical SOD2 nuclear-to-mitochondrial translocalization leading to overall ROS suppression. In a mouse xenograft metastasis model, the therapeutically relevant vitamin D derivative calcipotriol inhibited osteosarcoma metastasis and tumor growth shown for the first time. Our results uncover novel osteosarcoma-inhibiting mechanisms for vitamin D and calcipotriol that may be translated to human patients.
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31
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Zuniga G, Levy S, Ramirez P, Mange JD, Gonzalez E, Gamez M, Frost B. Tau-induced deficits in nonsense-mediated mRNA decay contribute to neurodegeneration. Alzheimers Dement 2023; 19:405-420. [PMID: 35416419 PMCID: PMC9673995 DOI: 10.1002/alz.12653] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2021] [Revised: 01/26/2022] [Accepted: 02/17/2022] [Indexed: 12/12/2022]
Abstract
INTRODUCTION While brains of patients with Alzheimer's disease and related tauopathies have evidence of altered RNA processing, we lack a mechanistic understanding of how altered RNA processing arises in these disorders and if such changes are causally linked to neurodegeneration. METHODS Using Drosophila melanogaster models of tauopathy, we find that overall activity of nonsense-mediated mRNA decay (NMD), a key RNA quality-control mechanism, is reduced. Genetic manipulation of NMD machinery significantly modifies tau-induced neurotoxicity, suggesting that deficits in NMD are causally linked to neurodegeneration. Mechanistically, we find that deficits in NMD are a consequence of aberrant RNA export and RNA accumulation within nuclear envelope invaginations in tauopathy. We identify a pharmacological activator of NMD that suppresses neurodegeneration in tau transgenic Drosophila, indicating that tau-induced deficits in RNA quality control are druggable. DISCUSSION Our studies suggest that NMD activators should be explored for their potential therapeutic value to patients with tauopathies.
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Affiliation(s)
- Gabrielle Zuniga
- Barshop Institute for Longevity and Aging Studies, University of Texas Health San Antonio, San Antonio, Texas
- Glenn Biggs Institute for Alzheimer’s and Neurodegenerative Diseases, University of Texas Health San Antonio, San Antonio, Texas
- Department of Cell Systems and Anatomy, University of Texas Health San Antonio, San Antonio, Texas
| | - Simon Levy
- Barshop Institute for Longevity and Aging Studies, University of Texas Health San Antonio, San Antonio, Texas
- Glenn Biggs Institute for Alzheimer’s and Neurodegenerative Diseases, University of Texas Health San Antonio, San Antonio, Texas
- Department of Cell Systems and Anatomy, University of Texas Health San Antonio, San Antonio, Texas
| | - Paulino Ramirez
- Barshop Institute for Longevity and Aging Studies, University of Texas Health San Antonio, San Antonio, Texas
- Glenn Biggs Institute for Alzheimer’s and Neurodegenerative Diseases, University of Texas Health San Antonio, San Antonio, Texas
- Department of Cell Systems and Anatomy, University of Texas Health San Antonio, San Antonio, Texas
| | - Jasmine De Mange
- Barshop Institute for Longevity and Aging Studies, University of Texas Health San Antonio, San Antonio, Texas
- Glenn Biggs Institute for Alzheimer’s and Neurodegenerative Diseases, University of Texas Health San Antonio, San Antonio, Texas
- Department of Cell Systems and Anatomy, University of Texas Health San Antonio, San Antonio, Texas
| | - Elias Gonzalez
- Barshop Institute for Longevity and Aging Studies, University of Texas Health San Antonio, San Antonio, Texas
- Glenn Biggs Institute for Alzheimer’s and Neurodegenerative Diseases, University of Texas Health San Antonio, San Antonio, Texas
- Department of Cell Systems and Anatomy, University of Texas Health San Antonio, San Antonio, Texas
| | - Maria Gamez
- Barshop Institute for Longevity and Aging Studies, University of Texas Health San Antonio, San Antonio, Texas
- Glenn Biggs Institute for Alzheimer’s and Neurodegenerative Diseases, University of Texas Health San Antonio, San Antonio, Texas
- Department of Cell Systems and Anatomy, University of Texas Health San Antonio, San Antonio, Texas
| | - Bess Frost
- Barshop Institute for Longevity and Aging Studies, University of Texas Health San Antonio, San Antonio, Texas
- Glenn Biggs Institute for Alzheimer’s and Neurodegenerative Diseases, University of Texas Health San Antonio, San Antonio, Texas
- Department of Cell Systems and Anatomy, University of Texas Health San Antonio, San Antonio, Texas
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Nonsense-Mediated mRNA Decay as a Mediator of Tumorigenesis. Genes (Basel) 2023; 14:genes14020357. [PMID: 36833284 PMCID: PMC9956241 DOI: 10.3390/genes14020357] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2022] [Revised: 01/23/2023] [Accepted: 01/26/2023] [Indexed: 02/03/2023] Open
Abstract
Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved and well-characterized biological mechanism that ensures the fidelity and regulation of gene expression. Initially, NMD was described as a cellular surveillance or quality control process to promote selective recognition and rapid degradation of erroneous transcripts harboring a premature translation-termination codon (PTC). As estimated, one-third of mutated and disease-causing mRNAs were reported to be targeted and degraded by NMD, suggesting the significance of this intricate mechanism in maintaining cellular integrity. It was later revealed that NMD also elicits down-regulation of many endogenous mRNAs without mutations (~10% of the human transcriptome). Therefore, NMD modulates gene expression to evade the generation of aberrant truncated proteins with detrimental functions, compromised activities, or dominant-negative effects, as well as by controlling the abundance of endogenous mRNAs. By regulating gene expression, NMD promotes diverse biological functions during development and differentiation, and facilitates cellular responses to adaptation, physiological changes, stresses, environmental insults, etc. Mutations or alterations (such as abnormal expression, degradation, post-translational modification, etc.) that impair the function or expression of proteins associated with the NMD pathway can be deleterious to cells and may cause pathological consequences, as implicated in developmental and intellectual disabilities, genetic defects, and cancer. Growing evidence in past decades has highlighted NMD as a critical driver of tumorigenesis. Advances in sequencing technologies provided the opportunity to identify many NMD substrate mRNAs in tumor samples compared to matched normal tissues. Interestingly, many of these changes are tumor-specific and are often fine-tuned in a tumor-specific manner, suggesting the complex regulation of NMD in cancer. Tumor cells differentially exploit NMD for survival benefits. Some tumors promote NMD to degrade a subset of mRNAs, such as those encoding tumor suppressors, stress response proteins, signaling proteins, RNA binding proteins, splicing factors, and immunogenic neoantigens. In contrast, some tumors suppress NMD to facilitate the expression of oncoproteins or other proteins beneficial for tumor growth and progression. In this review, we discuss how NMD is regulated as a critical mediator of oncogenesis to promote the development and progression of tumor cells. Understanding how NMD affects tumorigenesis differentially will pave the way for the development of more effective and less toxic, targeted therapeutic opportunities in the era of personalized medicine.
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de Oliveira Freitas Machado C, Schafranek M, Brüggemann M, Hernández Cañás M, Keller M, Di Liddo A, Brezski A, Blümel N, Arnold B, Bremm A, Wittig I, Jaé N, McNicoll F, Dimmeler S, Zarnack K, Müller-McNicoll M. Poison cassette exon splicing of SRSF6 regulates nuclear speckle dispersal and the response to hypoxia. Nucleic Acids Res 2023; 51:870-890. [PMID: 36620874 PMCID: PMC9881134 DOI: 10.1093/nar/gkac1225] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2022] [Revised: 12/06/2022] [Accepted: 12/10/2022] [Indexed: 01/10/2023] Open
Abstract
Hypoxia induces massive changes in alternative splicing (AS) to adapt cells to the lack of oxygen. Here, we identify the splicing factor SRSF6 as a key factor in the AS response to hypoxia. The SRSF6 level is strongly reduced in acute hypoxia, which serves a dual purpose: it allows for exon skipping and triggers the dispersal of nuclear speckles. Our data suggest that cells use dispersal of nuclear speckles to reprogram their gene expression during hypoxic adaptation and that SRSF6 plays an important role in cohesion of nuclear speckles. Down-regulation of SRSF6 is achieved through inclusion of a poison cassette exon (PCE) promoted by SRSF4. Removing the PCE 3' splice site using CRISPR/Cas9 abolishes SRSF6 reduction in hypoxia. Aberrantly high SRSF6 levels in hypoxia attenuate hypoxia-mediated AS and impair dispersal of nuclear speckles. As a consequence, proliferation and genomic instability are increased, while the stress response is suppressed. The SRSF4-PCE-SRSF6 hypoxia axis is active in different cancer types, and high SRSF6 expression in hypoxic tumors correlates with a poor prognosis. We propose that the ultra-conserved PCE of SRSF6 acts as a tumor suppressor and that its inclusion in hypoxia is crucial to reduce SRSF6 levels. This may prevent tumor cells from entering the metastatic route of hypoxia adaptation.
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Affiliation(s)
- Camila de Oliveira Freitas Machado
- Institute of Molecular Biosciences, Goethe University, Frankfurt am Main, Germany,Institute of Cardiovascular Regeneration, Goethe University, Frankfurt am Main, Germany
| | - Michal Schafranek
- Institute of Molecular Biosciences, Goethe University, Frankfurt am Main, Germany
| | - Mirko Brüggemann
- Institute of Molecular Biosciences, Goethe University, Frankfurt am Main, Germany,Buchmann Institute for Molecular Life Sciences (BMLS), Frankfurt am Main, Germany
| | | | - Mario Keller
- Institute of Molecular Biosciences, Goethe University, Frankfurt am Main, Germany,Buchmann Institute for Molecular Life Sciences (BMLS), Frankfurt am Main, Germany
| | - Antonella Di Liddo
- Buchmann Institute for Molecular Life Sciences (BMLS), Frankfurt am Main, Germany
| | - Andre Brezski
- Institute of Molecular Biosciences, Goethe University, Frankfurt am Main, Germany,Buchmann Institute for Molecular Life Sciences (BMLS), Frankfurt am Main, Germany
| | - Nicole Blümel
- Institute of Molecular Biosciences, Goethe University, Frankfurt am Main, Germany
| | - Benjamin Arnold
- Institute of Molecular Biosciences, Goethe University, Frankfurt am Main, Germany
| | - Anja Bremm
- Institute of Biochemistry II, Goethe University, Frankfurt am Main, Germany
| | - Ilka Wittig
- Functional Proteomics, Institute of Cardiovascular Physiology, Goethe University, Frankfurt am Main, Germany
| | - Nicolas Jaé
- Institute of Cardiovascular Regeneration, Goethe University, Frankfurt am Main, Germany
| | - François McNicoll
- Institute of Molecular Biosciences, Goethe University, Frankfurt am Main, Germany
| | - Stefanie Dimmeler
- Institute of Cardiovascular Regeneration, Goethe University, Frankfurt am Main, Germany
| | - Kathi Zarnack
- Correspondence may also be addressed to Kathi Zarnack.
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Palo A, Patel SA, Sahoo B, Chowdary TK, Dixit M. FRG1 is a direct transcriptional regulator of nonsense-mediated mRNA decay genes. Genomics 2023; 115:110539. [PMID: 36521634 DOI: 10.1016/j.ygeno.2022.110539] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2022] [Revised: 12/04/2022] [Accepted: 12/10/2022] [Indexed: 12/14/2022]
Abstract
FRG1 is the primary candidate gene for Fascioscapulohumeral Muscular Dystrophy. So far, its role has been reported in muscle development, vasculogenesis, angiogenesis, and tumorigenesis. Mechanistically studies suggest FRG1's role in RNA biogenesis which may have implications in multiple physiological processes and diseases, including tumorigenesis. Its probable role as hnRNP and association with NMD-related genes prompted us to look into FRG1's effect on NMD gene expression and the mechanism. Using microarray profiling in cell lines, we found that FRG1 altered the mRNA surveillance pathway and associated pathways, such as RNA transport and spliceosome machinery molecules. Multiple sequence alignment of core factors, namely, UPF1, UPF3B, and SMG1, showed conserved stretches of nucleotide sequence 'CTGGG'. Structural modeling followed by EMSA, ChIP-qPCR, and luciferase reporter assays showed 'CTGGG' as a FRG1 binding site. Analysis of the publicly available datasets showed that the expression of FRG1 correlates with NMD genes in different tissue types. We validated the effect of FRG1 on NMD gene transcription by qRT-PCR. Overall, FRG1 might be a transcriptional regulator of NMD genes.
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Affiliation(s)
- Ananya Palo
- National Institute of Science Education and Research, School of Biological Sciences, Bhubaneswar, Odisha 752050, India; Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai 400094, India
| | - Saket Awadhesbhai Patel
- National Institute of Science Education and Research, School of Biological Sciences, Bhubaneswar, Odisha 752050, India; Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai 400094, India
| | - Bibekananda Sahoo
- National Institute of Science Education and Research, School of Biological Sciences, Bhubaneswar, Odisha 752050, India; Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai 400094, India
| | - Tirumala Kumar Chowdary
- National Institute of Science Education and Research, School of Biological Sciences, Bhubaneswar, Odisha 752050, India; Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai 400094, India
| | - Manjusha Dixit
- National Institute of Science Education and Research, School of Biological Sciences, Bhubaneswar, Odisha 752050, India; Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai 400094, India.
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Chumchuen S, Sripichai O, Jearawiriyapaisarn N, Fucharoen S, Peerapittayamongkol C. Induction of fetal hemoglobin: Lentiviral shRNA knockdown of HBS1L in β0-thalassemia/HbE erythroid cells. PLoS One 2023; 18:e0281059. [PMID: 36888630 PMCID: PMC9994754 DOI: 10.1371/journal.pone.0281059] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2021] [Accepted: 01/16/2023] [Indexed: 03/09/2023] Open
Abstract
Imbalanced globin chain output contributes to thalassemia pathophysiology. Hence, induction of fetal hemoglobin in β-thalassemia and other β-hemoglobinopathies are of continuing interest for therapeutic approaches. Genome-wide association studies have identified three common genetic loci: namely β-globin (HBB), an intergenic region between MYB and HBS1L, and BCL11A underlying quantitative fetal hemoglobin production. Here, we report that knockdown of HBS1L (all known variants) using shRNA in early erythroblast obtained from β0-thalassemia/HbE patients triggers an upregulation of γ-globin mRNA 1.69 folds. There is modest perturbation of red cell differentiation assessed by flow cytometry and morphology studies. The levels of α- and β-globin mRNAs are relatively unaltered. Knockdown of HBS1L also increases the percentage of fetal hemoglobin around 16.7 folds when compared to non-targeting shRNA. Targeting HBS1L is attractive because of the potent induction of fetal hemoglobin and the modest effect on cell differentiation.
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Affiliation(s)
- Sukanya Chumchuen
- Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
| | - Orapan Sripichai
- Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand
| | - Natee Jearawiriyapaisarn
- Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand
| | - Suthat Fucharoen
- Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand
| | - Chayanon Peerapittayamongkol
- Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
- * E-mail: ,
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36
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Chousal JN, Sohni A, Vitting-Seerup K, Cho K, Kim M, Tan K, Porse B, Wilkinson MF, Cook-Andersen H. Progression of the pluripotent epiblast depends upon the NMD factor UPF2. Development 2022; 149:dev200764. [PMID: 36255229 PMCID: PMC9687065 DOI: 10.1242/dev.200764] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2022] [Accepted: 09/09/2022] [Indexed: 11/09/2022]
Abstract
Nonsense-mediated RNA decay (NMD) is a highly conserved RNA turnover pathway that degrades RNAs harboring in-frame stop codons in specific contexts. Loss of NMD factors leads to embryonic lethality in organisms spanning the phylogenetic scale, but the mechanism remains unknown. Here, we report that the core NMD factor, UPF2, is required for expansion of epiblast cells within the inner cell mass of mice in vivo. We identify NMD target mRNAs in mouse blastocysts - both canonical and alternatively processed mRNAs - including those encoding cell cycle arrest and apoptosis factors, raising the possibility that NMD is essential for embryonic cell proliferation and survival. In support, the inner cell mass of Upf2-null blastocysts rapidly regresses with outgrowth and is incompetent for embryonic stem cell derivation in vitro. In addition, we uncovered concordant temporal- and lineage-specific regulation of NMD factors and mRNA targets, indicative of a shift in NMD magnitude during peri-implantation development. Together, our results reveal developmental and molecular functions of the NMD pathway in the early embryo.
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Affiliation(s)
- Jennifer N. Chousal
- Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
- Department of Molecular Biology, University of California, San Diego, La Jolla, CA 92093, USA
| | - Abhishek Sohni
- Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Kristoffer Vitting-Seerup
- The Bioinformatics Centre, Department of Biology and Biotech Research & Innovation Centre, University of Copenhagen, 2200 Copenhagen, Denmark
- Section for Bioinformatics, Health Technology, Technical University of Denmark (DTU), 2800 Kongens Lyngby, Denmark
| | - Kyucheol Cho
- Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
- Department of Molecular Biology, University of California, San Diego, La Jolla, CA 92093, USA
| | - Matthew Kim
- Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Kun Tan
- Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Bo Porse
- The Finsen Laboratory, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, DK2200 Copenhagen, Denmark
- Biotech Research and Innovation Center (BRIC), University of Copenhagen, 2200 Copenhagen, Denmark
- Novo Nordisk Foundation Center for Stem Cell Biology, DanStem, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen, Denmark
| | - Miles F. Wilkinson
- Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
- Institute of Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Heidi Cook-Andersen
- Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
- Department of Molecular Biology, University of California, San Diego, La Jolla, CA 92093, USA
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Fonteneau G, Redding A, Hoag-Lee H, Sim ES, Heinrich S, Gaida MM, Grabocka E. Stress Granules Determine the Development of Obesity-Associated Pancreatic Cancer. Cancer Discov 2022; 12:1984-2005. [PMID: 35674408 PMCID: PMC9357213 DOI: 10.1158/2159-8290.cd-21-1672] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2021] [Revised: 05/25/2022] [Accepted: 06/06/2022] [Indexed: 02/07/2023]
Abstract
Obesity is a global epidemic and a major predisposing factor for cancer. Increasing evidence shows that obesity-associated stress is a key driver of cancer risk and progression. Previous work has identified the phase-separation organelles, stress granules (SG), as mutant KRAS-dependent mediators of stress adaptation. However, the dependence of tumorigenesis on these organelles is unknown. Here, we establish a causal link between SGs and pancreatic ductal adenocarcinoma (PDAC). Importantly, we uncover that dependence on SGs is drastically heightened in obesity-associated PDAC. Furthermore, we identify a previously unknown regulator and component of SGs, namely, the serine/arginine protein kinase 2 (SRPK2), as a specific determinant of SG formation in obesity-associated PDAC. We show that SRPK2-mediated SG formation in obesity-associated PDAC is driven by hyperactivation of the IGF1/PI3K/mTOR/S6K1 pathway and that S6K1 inhibition selectively attenuates SGs and impairs obesity-associated PDAC development. SIGNIFICANCE : We show that stress adaptation via the phase-separation organelles SGs mediates PDAC development. Moreover, preexisting stress conditions such as obesity are a driving force behind tumor SG dependence, and enhanced SG levels are key determinants and a chemopreventive target for obesity-associated PDAC. This article is highlighted in the In This Issue feature, p. 1825.
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Affiliation(s)
- Guillaume Fonteneau
- Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, 19107, USA
| | - Alexandra Redding
- Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, 19107, USA
| | - Hannah Hoag-Lee
- Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, 19107, USA
| | - Edward S. Sim
- Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, 19107, USA
- Current Address: University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Stefan Heinrich
- Department of Surgery, University Medical Center Mainz, JGU-Mainz, 55131 Mainz, Germany
| | - Matthias M. Gaida
- Institute of Pathology, University Medical Center Mainz, JGU-Mainz, 55131 Mainz, Germany
- Research Center for Immunotherapy, University Medical Center Mainz, JGU-Mainz, 55131 Mainz, Germany
- Joint Unit Immunopathology, Institute of Pathology, University Medical Center, JGU-Mainz and TRON, Translational Oncology at the University Medical Center, JGU-Mainz, 55131 Mainz, Germany
| | - Elda Grabocka
- Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, 19107, USA
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38
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Cho H, Abshire ET, Popp MW, Pröschel C, Schwartz JL, Yeo GW, Maquat LE. AKT constitutes a signal-promoted alternative exon-junction complex that regulates nonsense-mediated mRNA decay. Mol Cell 2022; 82:2779-2796.e10. [PMID: 35675814 PMCID: PMC9357146 DOI: 10.1016/j.molcel.2022.05.013] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2022] [Revised: 04/21/2022] [Accepted: 05/10/2022] [Indexed: 11/28/2022]
Abstract
Despite a long appreciation for the role of nonsense-mediated mRNA decay (NMD) in destroying faulty, disease-causing mRNAs and maintaining normal, physiologic mRNA abundance, additional effectors that regulate NMD activity in mammalian cells continue to be identified. Here, we describe a haploid-cell genetic screen for NMD effectors that has unexpectedly identified 13 proteins constituting the AKT signaling pathway. We show that AKT supersedes UPF2 in exon-junction complexes (EJCs) that are devoid of RNPS1 but contain CASC3, defining an unanticipated insulin-stimulated EJC. Without altering UPF1 RNA binding or ATPase activity, AKT-mediated phosphorylation of the UPF1 CH domain at T151 augments UPF1 helicase activity, which is critical for NMD and also decreases the dependence of helicase activity on ATP. We demonstrate that upregulation of AKT signaling contributes to the hyperactivation of NMD that typifies Fragile X syndrome, as exemplified using FMR1-KO neural stem cells derived from induced pluripotent stem cells.
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Affiliation(s)
- Hana Cho
- Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA; Center for RNA Biology, University of Rochester, Rochester, NY 14642, USA
| | - Elizabeth T Abshire
- Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA; Center for RNA Biology, University of Rochester, Rochester, NY 14642, USA
| | - Maximilian W Popp
- Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA; Center for RNA Biology, University of Rochester, Rochester, NY 14642, USA
| | - Christoph Pröschel
- Department of Biomedical Genetics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA; Stem Cell and Regenerative Medicine Institute, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA
| | - Joshua L Schwartz
- Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA; Stem Cell Program, University of California, San Diego, La Jolla, CA, USA; Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA, USA
| | - Gene W Yeo
- Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA; Stem Cell Program, University of California, San Diego, La Jolla, CA, USA; Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA, USA
| | - Lynne E Maquat
- Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA; Center for RNA Biology, University of Rochester, Rochester, NY 14642, USA.
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LncRNA-Profile-Based Screening of Extracellular Vesicles Released from Brain Endothelial Cells after Oxygen–Glucose Deprivation. Brain Sci 2022; 12:brainsci12081027. [PMID: 36009090 PMCID: PMC9405926 DOI: 10.3390/brainsci12081027] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2022] [Revised: 07/23/2022] [Accepted: 07/27/2022] [Indexed: 01/27/2023] Open
Abstract
Brain microvascular endothelial cells (BMECs) linked by tight junctions play important roles in cerebral ischemia. Intercellular signaling via extracellular vesicles (EVs) is an underappreciated mode of cell–cell crosstalk. This study aims to explore the potential function of long noncoding RNAs (lncRNAs) in BMECs’ secreted EVs. We subjected primary human and rat BMECs to oxygen and glucose deprivation (OGD). EVs were enriched for RNA sequencing. A comparison of the sequencing results revealed 146 upregulated lncRNAs and 331 downregulated lncRNAs in human cells and 1215 upregulated lncRNAs and 1200 downregulated lncRNAs in rat cells. Next, we analyzed the genes that were coexpressed with the differentially expressed (DE) lncRNAs on chromosomes and performed Gene Ontology (GO) and signaling pathway enrichment analyses. The results showed that the lncRNAs may play roles in apoptosis, the TNF signaling pathway, and leukocyte transendothelial migration. Next, three conserved lncRNAs between humans and rats were analyzed and confirmed using PCR. The binding proteins of these three lncRNAs in human astrocytes were identified via RNA pulldown and mass spectrometry. These proteins could regulate mRNA stability and translation. Additionally, the lentivirus was used to upregulate them in human microglial HMC3 cells. The results showed NR_002323.2 induced microglial M1 activation. Therefore, these results suggest that BMECs’ EVs carry the lncRNAs, which may regulate gliocyte function after cerebral ischemia.
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Tan K, Stupack DG, Wilkinson MF. Nonsense-mediated RNA decay: an emerging modulator of malignancy. Nat Rev Cancer 2022; 22:437-451. [PMID: 35624152 PMCID: PMC11009036 DOI: 10.1038/s41568-022-00481-2] [Citation(s) in RCA: 66] [Impact Index Per Article: 22.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 04/19/2022] [Indexed: 12/11/2022]
Abstract
Nonsense-mediated RNA decay (NMD) is a highly conserved RNA turnover pathway that selectively degrades RNAs harbouring truncating mutations that prematurely terminate translation, including nonsense, frameshift and some splice-site mutations. Recent studies show that NMD shapes the mutational landscape of tumours by selecting for mutations that tend to downregulate the expression of tumour suppressor genes but not oncogenes. This suggests that NMD can benefit tumours, a notion further supported by the finding that mRNAs encoding immunogenic neoantigen peptides are typically targeted for decay by NMD. Together, this raises the possibility that NMD-inhibitory therapy could be of therapeutic benefit against many tumour types, including those with a high load of neoantigen-generating mutations. Complicating this scenario is the evidence that NMD can also be detrimental for many tumour types, and consequently tumours often have perturbed NMD. NMD may suppress tumour generation and progression by degrading subsets of specific normal mRNAs, including those encoding stress-response proteins, signalling factors and other proteins beneficial for tumours, as well as pro-tumour non-coding RNAs. Together, these findings suggest that NMD-modulatory therapy has the potential to provide widespread therapeutic benefit against diverse tumour types. However, whether NMD should be stimulated or repressed requires careful analysis of the tumour to be treated.
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Affiliation(s)
- Kun Tan
- Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Diego, La Jolla, CA, USA
| | - Dwayne G Stupack
- Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Diego, La Jolla, CA, USA.
- UCSD Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA.
| | - Miles F Wilkinson
- Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Diego, La Jolla, CA, USA.
- Institute of Genomic Medicine, University of California, San Diego, La Jolla, CA, USA.
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Xiang Z, Liqing Y, Qingqing Y, Qiang H, Hongbo C. Retard or exacerbate: Role of long non-coding RNA growth arrest-specific 5 in the fibrosis. Cytokine Growth Factor Rev 2022; 67:89-104. [DOI: 10.1016/j.cytogfr.2022.06.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2022] [Revised: 06/10/2022] [Accepted: 06/13/2022] [Indexed: 11/26/2022]
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Bufton JC, Powers KT, Szeto JYA, Toelzer C, Berger I, Schaffitzel C. Structures of nonsense-mediated mRNA decay factors UPF3B and UPF3A in complex with UPF2 reveal molecular basis for competitive binding and for neurodevelopmental disorder-causing mutation. Nucleic Acids Res 2022; 50:5934-5947. [PMID: 35640974 PMCID: PMC9177958 DOI: 10.1093/nar/gkac421] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2022] [Revised: 05/01/2022] [Accepted: 05/10/2022] [Indexed: 11/14/2022] Open
Abstract
UPF3 is a key nonsense-mediated mRNA decay (NMD) factor required for mRNA surveillance and eukaryotic gene expression regulation. UPF3 exists as two paralogs (A and B) which are differentially expressed depending on cell type and developmental stage and believed to regulate NMD activity based on cellular requirements. UPF3B mutations cause intellectual disability. The underlying molecular mechanisms remain elusive, as many of the mutations lie in the poorly characterized middle-domain of UPF3B. Here, we show that UPF3A and UPF3B share structural and functional homology to paraspeckle proteins comprising an RNA-recognition motif-like domain (RRM-L), a NONA/paraspeckle-like domain (NOPS-L), and extended α-helical domain. These domains are essential for RNA/ribosome-binding, RNA-induced oligomerization and UPF2 interaction. Structures of UPF2's third middle-domain of eukaryotic initiation factor 4G (MIF4GIII) in complex with either UPF3B or UPF3A reveal unexpectedly intimate binding interfaces. UPF3B's disease-causing mutation Y160D in the NOPS-L domain displaces Y160 from a hydrophobic cleft in UPF2 reducing the binding affinity ∼40-fold compared to wildtype. UPF3A, which is upregulated in patients with the UPF3B-Y160D mutation, binds UPF2 with ∼10-fold higher affinity than UPF3B reliant mainly on NOPS-L residues. Our characterization of RNA- and UPF2-binding by UPF3's middle-domain elucidates its essential role in NMD.
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Affiliation(s)
- Joshua C Bufton
- School of Biochemistry, University of Bristol; University Walk, Bristol BS8 1TD, UK
| | - Kyle T Powers
- School of Biochemistry, University of Bristol; University Walk, Bristol BS8 1TD, UK
| | - Jenn-Yeu A Szeto
- School of Biochemistry, University of Bristol; University Walk, Bristol BS8 1TD, UK
| | - Christine Toelzer
- School of Biochemistry, University of Bristol; University Walk, Bristol BS8 1TD, UK
| | - Imre Berger
- School of Biochemistry, University of Bristol; University Walk, Bristol BS8 1TD, UK.,Max Planck Bristol Centre for Minimal Biology, Cantock's Close, Bristol BS8 1TS, UK
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Szewczyk MM, Luciani GM, Vu V, Murison A, Dilworth D, Barghout SH, Lupien M, Arrowsmith CH, Minden MD, Barsyte-Lovejoy D. PRMT5 regulates ATF4 transcript splicing and oxidative stress response. Redox Biol 2022; 51:102282. [PMID: 35305370 PMCID: PMC8933703 DOI: 10.1016/j.redox.2022.102282] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2022] [Revised: 02/18/2022] [Accepted: 03/08/2022] [Indexed: 02/07/2023] Open
Abstract
Protein methyltransferase 5 (PRMT5) symmetrically dimethylates arginine residues leading to regulation of transcription and splicing programs. Although PRMT5 has emerged as an attractive oncology target, the molecular determinants of PRMT5 dependency in cancer remain incompletely understood. Our transcriptomic analysis identified PRMT5 regulation of the activating transcription factor 4 (ATF4) pathway in acute myelogenous leukemia (AML). PRMT5 inhibition resulted in the expression of unstable, intron-retaining ATF4 mRNA that is detained in the nucleus. Concurrently, the decrease in the spliced cytoplasmic transcript of ATF4 led to lower levels of ATF4 protein and downregulation of ATF4 target genes. Upon loss of functional PRMT5, cells with low ATF4 displayed increased oxidative stress, growth arrest, and cellular senescence. Interestingly, leukemia cells with EVI1 oncogene overexpression demonstrated dependence on PRMT5 function. EVI1 and ATF4 regulated gene signatures were inversely correlated. We show that EVI1-high AML cells have reduced ATF4 levels, elevated baseline reactive oxygen species and increased sensitivity to PRMT5 inhibition. Thus, EVI1-high cells demonstrate dependence on PRMT5 function and regulation of oxidative stress response. Overall, our findings identify the PRMT5-ATF4 axis to be safeguarding the cellular redox balance that is especially important in high oxidative stress states, such as those that occur with EVI1 overexpression.
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Affiliation(s)
| | - Genna M Luciani
- Department of Medical Biophysics, University of Toronto, Ontario, Canada; Princess Margaret Cancer Centre, Toronto, Ontario, Canada
| | - Victoria Vu
- Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada; Department of Medical Biophysics, University of Toronto, Ontario, Canada
| | - Alex Murison
- Princess Margaret Cancer Centre, Toronto, Ontario, Canada
| | - David Dilworth
- Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada
| | - Samir H Barghout
- Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada
| | - Mathieu Lupien
- Department of Medical Biophysics, University of Toronto, Ontario, Canada; Princess Margaret Cancer Centre, Toronto, Ontario, Canada
| | - Cheryl H Arrowsmith
- Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada; Department of Medical Biophysics, University of Toronto, Ontario, Canada; Princess Margaret Cancer Centre, Toronto, Ontario, Canada
| | - Mark D Minden
- Department of Medical Biophysics, University of Toronto, Ontario, Canada; Princess Margaret Cancer Centre, Toronto, Ontario, Canada.
| | - Dalia Barsyte-Lovejoy
- Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada; Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, M5S 1A8, Canada.
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Zhao J, Li Z, Puri R, Liu K, Nunez I, Chen L, Zheng S. Molecular profiling of individual FDA-approved clinical drugs identifies modulators of nonsense-mediated mRNA decay. MOLECULAR THERAPY. NUCLEIC ACIDS 2022; 27:304-318. [PMID: 35024243 PMCID: PMC8718828 DOI: 10.1016/j.omtn.2021.12.003] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/12/2021] [Accepted: 12/07/2021] [Indexed: 12/12/2022]
Abstract
Nonsense-mediated mRNA decay (NMD) degrades transcripts with premature stop codons. Given the prevalence of nonsense single nucleotide polymorphisms (SNPs) in the general population, it is urgent to catalog the effects of clinically approved drugs on NMD activity: any interference could alter the expression of nonsense SNPs, inadvertently inducing adverse effects. This risk is higher for patients with disease-causing nonsense mutations or an illness linked to dysregulated nonsense transcripts. On the other hand, hundreds of disorders are affected by cellular NMD efficiency and may benefit from NMD-modulatory drugs. Here, we profiled individual FDA-approved drugs for their impact on cellular NMD efficiency using a sensitive method that directly probes multiple endogenous NMD targets for a robust readout of NMD modulation. We found most FDA-approved drugs cause unremarkable effects on NMD, while many elicit clear transcriptional responses. Besides several potential mild NMD modulators, the anticancer drug homoharringtonine (HHT or omacetaxine mepesuccinate) consistently upregulates various endogenous NMD substrates in a dose-dependent manner in multiple cell types. We further showed translation inhibition mediates HHT's NMD effect. In summary, many FDA drugs induce transcriptional changes, and a few impact global NMD, and direct measurement of endogenous NMD substrate expression is robust to monitor cellular NMD.
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Affiliation(s)
- Jingrong Zhao
- Division of Biomedical Sciences, School of Medicine, University of California, Riverside, CA 91709, USA
| | - Zhelin Li
- Division of Biomedical Sciences, School of Medicine, University of California, Riverside, CA 91709, USA
| | - Ruchira Puri
- Division of Biomedical Sciences, School of Medicine, University of California, Riverside, CA 91709, USA
| | - Kelvin Liu
- Division of Biomedical Sciences, School of Medicine, University of California, Riverside, CA 91709, USA
| | - Israel Nunez
- Division of Biomedical Sciences, School of Medicine, University of California, Riverside, CA 91709, USA
| | - Liang Chen
- Department of Quantitative and Computational Biology, University of Southern California, Los Angeles, CA 90089, USA
| | - Sika Zheng
- Division of Biomedical Sciences, School of Medicine, University of California, Riverside, CA 91709, USA
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Abstract
Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that eliminates transcripts containing premature termination codons (PTCs). Half-lives of the mRNAs containing PTCs demonstrate that a small percent escape surveillance and do not degrade. It is not known whether this escape represents variable mRNA degradation within cells or, alternatively cells within the population are resistant. Here we demonstrate a single-cell approach with a bi-directional reporter, which expresses two β-globin genes with or without a PTC in the same cell, to characterize the efficiency of NMD in individual cells. We found a broad range of NMD efficiency in the population; some cells degraded essentially all of the mRNAs, while others escaped NMD almost completely. Characterization of NMD efficiency together with NMD regulators in single cells showed cell-to-cell variability of NMD reflects the differential level of surveillance factors, SMG1 and phosphorylated UPF1. A single-cell fluorescent reporter system that enabled detection of NMD using flow cytometry revealed that this escape occurred either by translational readthrough at the PTC or by a failure of mRNA degradation after successful translation termination at the PTC.
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Bongiorno R, Colombo MP, Lecis D. Deciphering the nonsense-mediated mRNA decay pathway to identify cancer cell vulnerabilities for effective cancer therapy. J Exp Clin Cancer Res 2021; 40:376. [PMID: 34852841 PMCID: PMC8638473 DOI: 10.1186/s13046-021-02192-2] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2021] [Accepted: 11/22/2021] [Indexed: 12/22/2022] Open
Abstract
Nonsense-mediated mRNA decay (NMD) is a highly conserved cellular surveillance mechanism, commonly studied for its role in mRNA quality control because of its capacity of degrading mutated mRNAs that would produce truncated proteins. However, recent studies have proven that NMD hides more complex tasks involved in a plethora of cellular activities. Indeed, it can control the stability of mutated as well as non-mutated transcripts, tuning transcriptome regulation. NMD not only displays a pivotal role in cell physiology but also in a number of genetic diseases. In cancer, the activity of this pathway is extremely complex and it is endowed with both pro-tumor and tumor suppressor functions, likely depending on the genetic context and tumor microenvironment. NMD inhibition has been tested in pre-clinical studies showing favored production of neoantigens by cancer cells, which can stimulate the triggering of an anti-tumor immune response. At the same time, NMD inhibition could result in a pro-tumor effect, increasing cancer cell adaptation to stress. Since several NMD inhibitors are already available in the clinic to treat genetic diseases, these compounds could be redirected to treat cancer patients, pending the comprehension of these variegated NMD regulation mechanisms. Ideally, an effective strategy should exploit the anti-tumor advantages of NMD inhibition and simultaneously preserve its intrinsic tumor suppressor functions. The targeting of NMD could provide a new therapeutic opportunity, increasing the immunogenicity of tumors and potentially boosting the efficacy of the immunotherapy agents now available for cancer treatment.
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Affiliation(s)
- Roberta Bongiorno
- Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo 42, 20133, Milan, Italy
| | - Mario Paolo Colombo
- Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo 42, 20133, Milan, Italy
| | - Daniele Lecis
- Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo 42, 20133, Milan, Italy.
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Miles RR, Amin PH, Diaz MB, Misra J, Aukerman E, Das A, Ghosh N, Guith T, Knierman MD, Roy S, Spandau DF, Wek RC. The eIF2 kinase GCN2 directs keratinocyte collective cell migration during wound healing via coordination of reactive oxygen species and amino acids. J Biol Chem 2021; 297:101257. [PMID: 34597669 PMCID: PMC8554533 DOI: 10.1016/j.jbc.2021.101257] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2021] [Revised: 09/20/2021] [Accepted: 09/27/2021] [Indexed: 12/25/2022] Open
Abstract
Healing of cutaneous wounds requires the collective migration of epithelial keratinocytes to seal the wound bed from the environment. However, the signaling events that coordinate this collective migration are unclear. In this report, we address the role of phosphorylation of eukaryotic initiation factor 2 (eIF2) and attendant gene expression during wound healing. Wounding of human keratinocyte monolayers in vitro led to the rapid activation of the eIF2 kinase GCN2. We determined that deletion or pharmacological inhibition of GCN2 significantly delayed collective cell migration and wound closure. Global transcriptomic, biochemical, and cellular analyses indicated that GCN2 is necessary for maintenance of intracellular free amino acids, particularly cysteine, as well as coordination of RAC1-GTP-driven reactive oxygen species (ROS) generation, lamellipodia formation, and focal adhesion dynamics following keratinocyte wounding. In vivo experiments using mice deficient for GCN2 validated the role of the eIF2 kinase during wound healing in intact skin. These results indicate that GCN2 is critical for appropriate induction of collective cell migration and plays a critical role in coordinating the re-epithelialization of cutaneous wounds.
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Affiliation(s)
- Rebecca R Miles
- Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Parth H Amin
- Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Miguel Barriera Diaz
- Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Jagannath Misra
- Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Erica Aukerman
- Department of Dermatology, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Amitava Das
- Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana, USA; Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Nandini Ghosh
- Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana, USA; Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Tanner Guith
- Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana, USA; Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Michael D Knierman
- Laboratory for Experimental Medicine, Eli Lilly and Company, Indianapolis, Indiana, USA
| | - Sashwati Roy
- Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana, USA; Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Dan F Spandau
- Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA; Department of Dermatology, Indiana University School of Medicine, Indianapolis, Indiana, USA; Richard L. Roudebush Veterans Administration Medical Center, Indianapolis, Indiana, USA.
| | - Ronald C Wek
- Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
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Baudu T, Parratte C, Perez V, Ancion M, Millevoi S, Hervouet E, Peigney A, Peixoto P, Overs A, Herfs M, Fraichard A, Guittaut M, Baguet A. The NMD Pathway Regulates GABARAPL1 mRNA during the EMT. Biomedicines 2021; 9:biomedicines9101302. [PMID: 34680418 PMCID: PMC8533616 DOI: 10.3390/biomedicines9101302] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2021] [Revised: 09/14/2021] [Accepted: 09/17/2021] [Indexed: 11/23/2022] Open
Abstract
EMT is a reversible cellular process that is linked to gene expression reprogramming, which allows for epithelial cells to undergo a phenotypic switch to acquire mesenchymal properties. EMT is associated with cancer progression and cancer therapeutic resistance and it is known that, during the EMT, many stress response pathways, such as autophagy and NMD, are dysregulated. Therefore, our goal was to study the regulation of ATG8 family members (GABARAP, GABARAPL1, LC3B) by the NMD and to identify molecular links between these two cellular processes that are involved in tumor development and metastasis formation. IHC experiments, which were conducted in a cohort of patients presenting lung adenocarcinomas, showed high GABARAPL1 and low UPF1 levels in EMT+ tumors. We observed increased levels of GABARAPL1 correlated with decreased levels of NMD factors in A549 cells in vitro. We then confirmed that GABARAPL1 mRNA was indeed targeted by the NMD in a 3′UTR-dependent manner and we identified four overlapping binding sites for UPF1 and eIF4A3 that are potentially involved in the recognition of this transcript by the NMD pathway. Our study suggests that 3′UTR-dependent NMD might be an important mechanism that is involved in the induction of autophagy and could represent a promising target in the development of new anti-cancer therapies.
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Affiliation(s)
- Timothée Baudu
- INSERM, EFS BFC, UMR1098, RIGHT Institute, Interactions Hôte-Greffon-Tumeur/Ingénierie Cellulaire et Génique, University Bourgogne Franche-Comté, 25000 Besançon, France; (T.B.); (C.P.); (V.P.); (E.H.); (A.P.); (P.P.); (A.O.); (A.F.); (M.G.)
| | - Chloé Parratte
- INSERM, EFS BFC, UMR1098, RIGHT Institute, Interactions Hôte-Greffon-Tumeur/Ingénierie Cellulaire et Génique, University Bourgogne Franche-Comté, 25000 Besançon, France; (T.B.); (C.P.); (V.P.); (E.H.); (A.P.); (P.P.); (A.O.); (A.F.); (M.G.)
| | - Valérie Perez
- INSERM, EFS BFC, UMR1098, RIGHT Institute, Interactions Hôte-Greffon-Tumeur/Ingénierie Cellulaire et Génique, University Bourgogne Franche-Comté, 25000 Besançon, France; (T.B.); (C.P.); (V.P.); (E.H.); (A.P.); (P.P.); (A.O.); (A.F.); (M.G.)
| | - Marie Ancion
- Laboratory of Experimental Pathology, GIGA-Cancer, University of Liege, B-4000 Liege, Belgium; (M.A.); (M.H.)
| | - Stefania Millevoi
- Cancer Research Centre of Toulouse, INSERM UMR 1037, Université Toulouse III-Paul Sabatier, 31330 Toulouse, France;
| | - Eric Hervouet
- INSERM, EFS BFC, UMR1098, RIGHT Institute, Interactions Hôte-Greffon-Tumeur/Ingénierie Cellulaire et Génique, University Bourgogne Franche-Comté, 25000 Besançon, France; (T.B.); (C.P.); (V.P.); (E.H.); (A.P.); (P.P.); (A.O.); (A.F.); (M.G.)
- DImaCell platform, University Bourgogne Franche-Comté, 25000 Besançon, France
- EPIGENEXP platform, University Bourgogne Franche-Comté, 25000 Besançon, France
| | - Anne Peigney
- INSERM, EFS BFC, UMR1098, RIGHT Institute, Interactions Hôte-Greffon-Tumeur/Ingénierie Cellulaire et Génique, University Bourgogne Franche-Comté, 25000 Besançon, France; (T.B.); (C.P.); (V.P.); (E.H.); (A.P.); (P.P.); (A.O.); (A.F.); (M.G.)
- EPIGENEXP platform, University Bourgogne Franche-Comté, 25000 Besançon, France
| | - Paul Peixoto
- INSERM, EFS BFC, UMR1098, RIGHT Institute, Interactions Hôte-Greffon-Tumeur/Ingénierie Cellulaire et Génique, University Bourgogne Franche-Comté, 25000 Besançon, France; (T.B.); (C.P.); (V.P.); (E.H.); (A.P.); (P.P.); (A.O.); (A.F.); (M.G.)
- EPIGENEXP platform, University Bourgogne Franche-Comté, 25000 Besançon, France
| | - Alexis Overs
- INSERM, EFS BFC, UMR1098, RIGHT Institute, Interactions Hôte-Greffon-Tumeur/Ingénierie Cellulaire et Génique, University Bourgogne Franche-Comté, 25000 Besançon, France; (T.B.); (C.P.); (V.P.); (E.H.); (A.P.); (P.P.); (A.O.); (A.F.); (M.G.)
- Laboratoire de Biochimie, CHU Besançon, 25000 Besançon, France
| | - Michael Herfs
- Laboratory of Experimental Pathology, GIGA-Cancer, University of Liege, B-4000 Liege, Belgium; (M.A.); (M.H.)
| | - Annick Fraichard
- INSERM, EFS BFC, UMR1098, RIGHT Institute, Interactions Hôte-Greffon-Tumeur/Ingénierie Cellulaire et Génique, University Bourgogne Franche-Comté, 25000 Besançon, France; (T.B.); (C.P.); (V.P.); (E.H.); (A.P.); (P.P.); (A.O.); (A.F.); (M.G.)
| | - Michaël Guittaut
- INSERM, EFS BFC, UMR1098, RIGHT Institute, Interactions Hôte-Greffon-Tumeur/Ingénierie Cellulaire et Génique, University Bourgogne Franche-Comté, 25000 Besançon, France; (T.B.); (C.P.); (V.P.); (E.H.); (A.P.); (P.P.); (A.O.); (A.F.); (M.G.)
- DImaCell platform, University Bourgogne Franche-Comté, 25000 Besançon, France
| | - Aurélie Baguet
- INSERM, EFS BFC, UMR1098, RIGHT Institute, Interactions Hôte-Greffon-Tumeur/Ingénierie Cellulaire et Génique, University Bourgogne Franche-Comté, 25000 Besançon, France; (T.B.); (C.P.); (V.P.); (E.H.); (A.P.); (P.P.); (A.O.); (A.F.); (M.G.)
- DImaCell platform, University Bourgogne Franche-Comté, 25000 Besançon, France
- Correspondence:
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Silva J, Nina P, Romão L. Translation of ABCE1 Is Tightly Regulated by Upstream Open Reading Frames in Human Colorectal Cells. Biomedicines 2021; 9:biomedicines9080911. [PMID: 34440115 PMCID: PMC8389594 DOI: 10.3390/biomedicines9080911] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2021] [Accepted: 07/26/2021] [Indexed: 11/29/2022] Open
Abstract
ATP-binding cassette subfamily E member 1 (ABCE1) belongs to the ABC protein family of transporters; however, it does not behave as a drug transporter. Instead, ABCE1 actively participates in different stages of translation and is also associated with oncogenic functions. Ribosome profiling analysis in colorectal cancer cells has revealed a high ribosome occupancy in the human ABCE1 mRNA 5′-leader sequence, indicating the presence of translatable upstream open reading frames (uORFs). These cis-acting translational regulatory elements usually act as repressors of translation of the main coding sequence. In the present study, we dissect the regulatory function of the five AUG and five non-AUG uORFs identified in the human ABCE1 mRNA 5′-leader sequence. We show that the expression of the main coding sequence is tightly regulated by the ABCE1 AUG uORFs in colorectal cells. Our results are consistent with a model wherein uORF1 is efficiently translated, behaving as a barrier to downstream uORF translation. The few ribosomes that can bypass uORF1 (and/or uORF2) must probably initiate at the inhibitory uORF3 or uORF5 that efficiently repress translation of the main ORF. This inhibitory property is slightly overcome in conditions of endoplasmic reticulum stress. In addition, we observed that these potent translation-inhibitory AUG uORFs function equally in cancer and in non-tumorigenic colorectal cells, which is consistent with a lack of oncogenic function. In conclusion, we establish human ABCE1 as an additional example of uORF-mediated translational regulation and that this tight regulation contributes to control ABCE1 protein levels in different cell environments.
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Affiliation(s)
- Joana Silva
- Departamento de Genética Humana, Instituto Nacional de Saúde Doutor Ricardo Jorge, 1649-016 Lisboa, Portugal; (J.S.); (P.N.)
- Instituto de Biossistemas e Ciências Integrativas (BioISI), Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal
| | - Pedro Nina
- Departamento de Genética Humana, Instituto Nacional de Saúde Doutor Ricardo Jorge, 1649-016 Lisboa, Portugal; (J.S.); (P.N.)
| | - Luísa Romão
- Departamento de Genética Humana, Instituto Nacional de Saúde Doutor Ricardo Jorge, 1649-016 Lisboa, Portugal; (J.S.); (P.N.)
- Instituto de Biossistemas e Ciências Integrativas (BioISI), Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal
- Correspondence: ; Tel.: +351-21-750-8155
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50
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Andjus S, Morillon A, Wery M. From Yeast to Mammals, the Nonsense-Mediated mRNA Decay as a Master Regulator of Long Non-Coding RNAs Functional Trajectory. Noncoding RNA 2021; 7:ncrna7030044. [PMID: 34449682 PMCID: PMC8395947 DOI: 10.3390/ncrna7030044] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2021] [Revised: 07/22/2021] [Accepted: 07/25/2021] [Indexed: 12/22/2022] Open
Abstract
The Nonsense-Mediated mRNA Decay (NMD) has been classically viewed as a translation-dependent RNA surveillance pathway degrading aberrant mRNAs containing premature stop codons. However, it is now clear that mRNA quality control represents only one face of the multiple functions of NMD. Indeed, NMD also regulates the physiological expression of normal mRNAs, and more surprisingly, of long non-coding (lnc)RNAs. Here, we review the different mechanisms of NMD activation in yeast and mammals, and we discuss the molecular bases of the NMD sensitivity of lncRNAs, considering the functional roles of NMD and of translation in the metabolism of these transcripts. In this regard, we describe several examples of functional micropeptides produced from lncRNAs. We propose that translation and NMD provide potent means to regulate the expression of lncRNAs, which might be critical for the cell to respond to environmental changes.
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Affiliation(s)
- Sara Andjus
- ncRNA, Epigenetic and Genome Fluidity, Institut Curie, PSL University, Sorbonne Université, CNRS UMR3244, 26 Rue d’Ulm, CEDEX 05, F-75248 Paris, France;
| | - Antonin Morillon
- ncRNA, Epigenetic and Genome Fluidity, Institut Curie, Sorbonne Université, CNRS UMR3244, 26 Rue d’Ulm, CEDEX 05, F-75248 Paris, France
- Correspondence: (A.M.); (M.W.)
| | - Maxime Wery
- ncRNA, Epigenetic and Genome Fluidity, Institut Curie, Sorbonne Université, CNRS UMR3244, 26 Rue d’Ulm, CEDEX 05, F-75248 Paris, France
- Correspondence: (A.M.); (M.W.)
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