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Sterrett MC, Cureton LA, Cohen LN, van Hoof A, Khoshnevis S, Fasken MB, Corbett AH, Ghalei H. Comparative analyses of disease-linked missense mutations in the RNA exosome modeled in budding yeast reveal distinct functional consequences in translation. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2023.10.18.562946. [PMID: 37904946 PMCID: PMC10614903 DOI: 10.1101/2023.10.18.562946] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/02/2023]
Abstract
The RNA exosome is a multi-subunit, evolutionarily conserved ribonuclease complex that is essential for processing, decay and surveillance of many cellular RNAs. Missense mutations in genes encoding the structural subunits of the RNA exosome complex cause a diverse range of diseases, collectively known as RNA exosomopathies, often involving neurological and developmental defects. The varied symptoms suggest that different mutations lead to distinct in vivo consequences. To investigate these functional consequences and distinguish whether they are unique to each RNA exosomopathy mutation, we generated a collection of in vivo models by introducing pathogenic missense mutations in orthologous S. cerevisiae genes. Comparative RNA-seq analysis assessing broad transcriptomic changes in each mutant model revealed that three yeast mutant models, rrp4-G226D, rrp40-W195R and rrp46-L191H, which model mutations in the genes encoding EXOSC2, EXOSC3 and EXOSC5, respectively, had the largest transcriptomic differences. While some transcriptomic changes, particularly in transcripts related to ribosome biogenesis, were shared among mutant models, each mutation also induced unique transcriptomic changes. Thus, our data suggests that while there are some shared consequences, there are also distinct differences in RNA exosome function by each variant. Assessment of ribosome biogenesis and translation defects in the three models revealed distinct differences in polysome profiles. Collectively, our results provide the first comparative analyses of RNA exosomopathy mutant models and suggest that different RNA exosome gene mutations result in in vivo consequences that are both unique and shared across each variant, providing further insight into the biology underlying each distinct pathology.
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Affiliation(s)
- Maria C. Sterrett
- Department of Biology, Emory University, Atlanta, Georgia, USA
- Biochemistry, Cell and Developmental Biology Graduate Program, Emory University, Atlanta, Georgia, USA
| | - Lauryn A. Cureton
- Genetics and Molecular Biology Graduate Program, Emory University, Atlanta, Georgia, USA
- Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Lauren N. Cohen
- Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Ambro van Hoof
- Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center at Houston, Houston, Texas, USA
| | - Sohail Khoshnevis
- Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Milo B. Fasken
- Department of Biology, Emory University, Atlanta, Georgia, USA
| | | | - Homa Ghalei
- Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA
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Hurtig JE, Stuart CJ, van Hoof A. Independent neofunctionalization of Dxo1 in Saccharomyces and Candida led to 25S rRNA processing function. RNA (NEW YORK, N.Y.) 2024; 30:1634-1645. [PMID: 39332835 PMCID: PMC11571810 DOI: 10.1261/rna.080210.124] [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: 07/30/2024] [Accepted: 09/16/2024] [Indexed: 09/29/2024]
Abstract
Eukaryotic genomes typically encode one member of the DXO/Dxo1/Rai1 family of enzymes, which can hydrolyze the 5' ends of RNAs with a variety of structures that deviate from the canonical 7mGpppN. In contrast, the Saccharomyces genome encodes two family members and the second copy, Dxo1, is a distributive 5' exoribonuclease that is required for the final maturation of the 5' end of 25S rRNA from a 25S' precursor. Here we show that this 25S rRNA maturation function is not conserved across kingdoms, but arose in the budding yeasts. Interestingly, the origin of 25S processing capacity coincides with the duplication of this gene, and this capacity is absent in the nonduplicated genes. Strikingly, two different clades of budding yeasts have undergone parallel evolution: Both duplicated their DXO/Dxo1/Rai1 gene, and in both cases, one copy gained the 25S processing function. This was accompanied by many parallel sequence changes, a remarkable case of reproducible neofunctionalization.
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Affiliation(s)
- Jennifer E Hurtig
- Department of Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA
- UT MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA
| | - Catherine J Stuart
- Department of Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA
| | - Ambro van Hoof
- Department of Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA
- UT MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA
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3
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Wang L, Chen B, Ma B, Wang Y, Wang H, Sun X, Tan BC. Maize Dek51 encodes a DEAD-box RNA helicase essential for pre-rRNA processing and seed development. Cell Rep 2024; 43:114673. [PMID: 39196780 DOI: 10.1016/j.celrep.2024.114673] [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: 05/13/2024] [Revised: 07/09/2024] [Accepted: 08/08/2024] [Indexed: 08/30/2024] Open
Abstract
Pre-rRNA processing is essential to ribosome biosynthesis. However, the processing mechanism is not fully understood in plants. Here, we report a DEAD-box RNA helicase DEK51 that mediates the 3' end processing of 18S and 5.8S pre-rRNA in maize (Zea mays L.). DEK51 is localized in the nucleolus, and loss of DEK51 arrests maize seed development and blocks the 3' end processing of 18S and 5.8S pre-rRNA. DEK51 interacts with putative key factors in nuclear RNA exosome-mediated pre-rRNA processing, including ZmMTR4, ZmSMO4, ZmRRP44A, and ZmRRP6L2. This suggests that DEK51 facilitates pre-rRNA processing by interacting with the exosome. Loss of ZmMTR4 function arrests seed development and blocks the 3' end processing of 18S and 5.8S pre-rRNA, similar to dek51. DEK51 also interacts with endonucleases ZmUTP24 and ZmRCL1, suggesting that it may also be involved in the cleavage at site A2. These results show the critical role of DEK51 in promoting 3' end processing of pre-rRNA.
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Affiliation(s)
- Le Wang
- Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266237, China
| | - Baoyin Chen
- Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266237, China
| | - Bing Ma
- Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266237, China
| | - Yong Wang
- Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266237, China
| | - Hongqiu Wang
- National Key Laboratory of Wheat and Maize Crop Science, Collaborative Innovation Center of Henan Grain Crops, Henan Agricultural University, Zhengzhou 450002, China
| | - Xiaotong Sun
- Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266237, China
| | - Bao-Cai Tan
- Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266237, China.
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Zhang W, Cao Y, Li H, Rasmey AHM, Zhang K, Shi L, Ge B. Membrane protein Bcsdr2 mediates biofilm integrity, hyphal growth and virulence of Botrytis cinerea. Appl Microbiol Biotechnol 2024; 108:398. [PMID: 38940906 PMCID: PMC11213811 DOI: 10.1007/s00253-024-13238-8] [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: 01/13/2024] [Revised: 06/10/2024] [Accepted: 06/14/2024] [Indexed: 06/29/2024]
Abstract
Grey mould caused by Botrytis cinerea is a devastating disease responsible for large losses to agricultural production, and B. cinerea is a necrotrophic model fungal plant pathogen. Membrane proteins are important targets of fungicides and hotspots in the research and development of fungicide products. Wuyiencin affects the permeability and pathogenicity of B. cinerea, parallel reaction monitoring revealed the association of membrane protein Bcsdr2, and the bacteriostatic mechanism of wuyiencin was elucidated. In the present work, we generated and characterised ΔBcsdr2 deletion and complemented mutant B. cinerea strains. The ΔBcsdr2 deletion mutants exhibited biofilm loss and dissolution, and their functional activity was illustrated by reduced necrotic colonisation on strawberry and grape fruits. Targeted deletion of Bcsdr2 also blocked several phenotypic defects in aspects of mycelial growth, conidiation and virulence. All phenotypic defects were restored by targeted gene complementation. The roles of Bcsdr2 in biofilms and pathogenicity were also supported by quantitative real-time RT-PCR results showing that phosphatidylserine decarboxylase synthesis gene Bcpsd and chitin synthase gene BcCHSV II were downregulated in the early stages of infection for the ΔBcsdr2 strain. The results suggest that Bcsdr2 plays important roles in regulating various cellular processes in B. cinerea. KEY POINTS: • The mechanism of wuyiencin inhibits B. cinerea is closely associated with membrane proteins. • Wuyiencin can downregulate the expression of the membrane protein Bcsdr2 in B. cinerea. • Bcsdr2 is involved in regulating B. cinerea virulence, growth and development.
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Affiliation(s)
- Wei Zhang
- State Key Laboratory of Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 2 Yuanmingyuan West Road, Beijing, 100193, China
- Qian Xinan Branch of Guizhou Provincial Tobacco Company, 60 Ruijin Southern Road, Xingyi, 562499, China
| | - Yi Cao
- Guizhou Academy of Tobacco Science, 29 Longtanba Road, Guiyang, 550081, China
| | - Hua Li
- School of Light Industry Science and Engineering, Beijing Technology and Business University, 11 & 33 Fucheng Road, Beijing, 100048, China
| | - Abdel-Hamied M Rasmey
- Botany and Microbiology Department, Faculty of Science, Suez University, Elsalam 1, Cairo-Suez Road, Suez, 43221, Egypt
| | - Kecheng Zhang
- State Key Laboratory of Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 2 Yuanmingyuan West Road, Beijing, 100193, China
| | - Liming Shi
- State Key Laboratory of Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 2 Yuanmingyuan West Road, Beijing, 100193, China.
| | - Beibei Ge
- State Key Laboratory of Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 2 Yuanmingyuan West Road, Beijing, 100193, China.
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Gerhalter M, Kofler L, Zisser G, Merl-Pham J, Hauck SM, Bergler H. The novel pre-rRNA detection workflow "Riboprobing" allows simple identification of undescribed RNA species. RNA (NEW YORK, N.Y.) 2024; 30:807-823. [PMID: 38580456 PMCID: PMC11182013 DOI: 10.1261/rna.079912.123] [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: 12/05/2023] [Accepted: 03/16/2024] [Indexed: 04/07/2024]
Abstract
Ribosomes translate mRNA into proteins and are essential for every living organism. In eukaryotes, both ribosomal subunits are rapidly assembled in a strict hierarchical order, starting in the nucleolus with the transcription of a common precursor ribosomal RNA (pre-rRNA). This pre-rRNA encodes three of the four mature rRNAs, which are formed by several, consecutive endonucleolytic and exonucleolytic processing steps. Historically, northern blots are used to analyze the variety of different pre-rRNA species, only allowing rough length estimations. Although this limitation can be overcome with primer extension, both approaches often use radioactivity and are time-consuming and costly. Here, we present "Riboprobing," a linker ligation-based workflow followed by reverse transcription and PCR for easy and fast detection and characterization of pre-rRNA species and their 5' as well as 3' ends. Using standard molecular biology laboratory equipment, "Riboprobing" allows reliable discrimination of pre-rRNA species not resolved by northern blot (e.g., 27SA2, 27SA3, and 27SB pre-rRNA). The method can successfully be used for the analysis of total cell extracts as well as purified pre-ribosomes for a straightforward evaluation of the impact of mutant gene versions or inhibitors. In the course of method development, we identified and characterized a hitherto undescribed aberrant pre-rRNA arising from LiCl inhibition. This pre-rRNA fragment spans from processing site A1 to E, forming a small RNP that lacks most early joining assembly factors. This finding expands our knowledge of how the cell deals with severe pre-rRNA processing defects and demonstrates the strict requirement for the 5'ETS (external transcribed spacer) for the assembly process.
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Affiliation(s)
| | - Lisa Kofler
- Institute of Molecular Biosciences, University of Graz, Graz 8010, Austria
| | - Gertrude Zisser
- Institute of Molecular Biosciences, University of Graz, Graz 8010, Austria
| | - Juliane Merl-Pham
- Metabolomics and Proteomics Core, Helmholtz Center Munich, Munich 80939, Germany
| | - Stefanie M Hauck
- Metabolomics and Proteomics Core, Helmholtz Center Munich, Munich 80939, Germany
| | - Helmut Bergler
- Institute of Molecular Biosciences, University of Graz, Graz 8010, Austria
- BioTechMed-Graz, Graz 8010, Austria
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Zhou H, Yuan W, Lei W, Zhou T, Qin P, Zhang B, Hu M. Domain definition and preliminary functional exploration of the endonuclease NOBP-1 in Strongyloides stercoralis. Parasit Vectors 2023; 16:399. [PMID: 37924155 PMCID: PMC10623843 DOI: 10.1186/s13071-023-05940-9] [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: 06/19/2023] [Accepted: 08/22/2023] [Indexed: 11/06/2023] Open
Abstract
BACKGROUND Ribosome biogenesis is the process of assembling ribosome complexes that regulate cell proliferation and differentiation with potential regulatory effects on development. Many factors regulate ribosome biological processes. Nin one binding protein (Nob1) has received widespread attention as key genes regulating ribosome biogenesis-the 3' end of the 20S rRNA is cleaved by Nob1 at cleavage site D to form 18S rRNA, generating translationally capable 40S subunit. As a ribosome biogenesis factor, Nob1 may regulate the development of organisms, but almost nothing is known about the function of Nob1 for any parasitic nematode. We explored the functional role of NOBP-1 (the homologous gene of Nob1) encoding gene from a parasitic nematode-Strongyloides stercoralis. METHODS The full-length cDNA, gDNA and promoter region of Ss-nobp-1 was identified using protein BLAST in WormBase ParaSite according to the Caenorhabditis elegans NOBP-1 sequence to analyze the gene structure. RNA sequencing (RNA-seq) data in wormbase were retrieved and analyzed to assess the transcript abundance of Ss-nobp-1 in seven developmental stages of S. stercoralis. The standard method for gonadal microinjection of constructs was carried out to determine the anatomic expression patterns of Ss-nobp-1. The interaction between Ss-NOBP-1 and partner of NOBP-1 (Ss-PNO-1) was assessed by yeast two-hybridization and bimolecular fluorescence complementarity (BiFC) experiments. RESULTS The NOBP-1 encoding gene Ss-nopb-1 from the zoonotic parasite S. stercoralis has been isolated and characterized. The genomic DNA representing Ss-nobp-1 includes a 1599-bp coding region and encodes a protein comprising 403 amino acids (aa), which contains conserved PIN domain and zinc ribbon domain. RNA-seq analysis revealed that Ss-nobp-1 transcripts are present throughout the seven developmental stages in S. stercoralis and have higher transcription levels in iL3, L3 and P Female. Ss-nobp-1 is expressed mainly in the intestine of transgenic S. stercoralis larvae, and there is a direct interaction between Ss-NOBP-1 and Ss-PNO-1. CONCLUSIONS Collectively, Ss-NOBP-1 has a potential role in embryo formation and the infective process, and findings from this study provide a sound foundation for investigating its function during the development of parasitic nematode.
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Affiliation(s)
- Huan Zhou
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
- National Key Laboratory of Cotton Bio-Breeding and Integrated Utilization, School of Life Sciences, Henan University, Kaifeng, China.
| | - Wang Yuan
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China
| | - Weiqiang Lei
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China
- College of Animal Science and Technology, Jinling Institute of Technology, Nanjing, 210038, China
| | - Taoxun Zhou
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China
| | - Peixi Qin
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China
| | - Biying Zhang
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China
| | - Min Hu
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
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Wolters SM, Benninghaus VA, Roelfs KU, van Deenen N, Twyman RM, Prüfer D, Schulze Gronover C. Overexpression of a pseudo-etiolated-in-light-like protein in Taraxacum koksaghyz leads to a pale green phenotype and enables transcriptome-based network analysis of photomorphogenesis and isoprenoid biosynthesis. FRONTIERS IN PLANT SCIENCE 2023; 14:1228961. [PMID: 37841614 PMCID: PMC10569127 DOI: 10.3389/fpls.2023.1228961] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 05/30/2023] [Accepted: 08/21/2023] [Indexed: 10/17/2023]
Abstract
Introduction Plant growth and greening in response to light require the synthesis of photosynthetic pigments such as chlorophylls and carotenoids, which are derived from isoprenoid precursors. In Arabidopsis, the pseudo-etiolated-in-light phenotype is caused by the overexpression of repressor of photosynthetic genes 2 (RPGE2), which regulates chlorophyll synthesis and photosynthetic genes. Methods We investigated a homologous protein in the Russian dandelion (Taraxacum koksaghyz) to determine its influence on the rich isoprenoid network in this species, using a combination of in silico analysis, gene overexpression, transcriptomics and metabolic profiling. Results Homology-based screening revealed a gene designated pseudo-etiolated-in-light-like (TkPEL-like), and in silico analysis identified a light-responsive G-box element in its promoter. TkPEL-like overexpression in dandelion plants and other systems reduced the levels of chlorophylls and carotenoids, but this was ameliorated by the mutation of one or both conserved cysteine residues. Comparative transcriptomics in dandelions overexpressing TkPEL-like showed that genes responsible for the synthesis of isoprenoid precursors and chlorophyll were downregulated, probably explaining the observed pale green leaf phenotype. In contrast, genes responsible for carotenoid synthesis were upregulated, possibly in response to feedback signaling. The evaluation of additional differentially expressed genes revealed interactions between pathways. Discussion We propose that TkPEL-like negatively regulates chlorophyll- and photosynthesis-related genes in a light-dependent manner, which appears to be conserved across species. Our data will inform future studies addressing the regulation of leaf isoprenoid biosynthesis and photomorphogenesis and could be used in future breeding strategies to optimize selected plant isoprenoid profiles and generate suitable plant-based production platforms.
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Affiliation(s)
- Silva Melissa Wolters
- Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Münster, Germany
| | | | - Kai-Uwe Roelfs
- Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Münster, Germany
| | - Nicole van Deenen
- Institute for Biology and Biotechnology of Plants, University of Münster, Münster, Germany
| | | | - Dirk Prüfer
- Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Münster, Germany
- Institute for Biology and Biotechnology of Plants, University of Münster, Münster, Germany
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Schneider C, Bohnsack KE. Caught in the act-Visualizing ribonucleases during eukaryotic ribosome assembly. WILEY INTERDISCIPLINARY REVIEWS. RNA 2023; 14:e1766. [PMID: 36254602 DOI: 10.1002/wrna.1766] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/14/2022] [Revised: 09/22/2022] [Accepted: 09/24/2022] [Indexed: 07/20/2023]
Abstract
Ribosomes are essential macromolecular machines responsible for translating the genetic information encoded in mRNAs into proteins. Ribosomes are composed of ribosomal RNAs and proteins (rRNAs and RPs) and the rRNAs fulfill both catalytic and architectural functions. Excision of the mature eukaryotic rRNAs from their precursor transcript is achieved through a complex series of endoribonucleolytic cleavages and exoribonucleolytic processing steps that are precisely coordinated with other aspects of ribosome assembly. Many ribonucleases involved in pre-rRNA processing have been identified and pre-rRNA processing pathways are relatively well defined. However, momentous advances in cryo-electron microscopy have recently enabled structural snapshots of various pre-ribosomal particles from budding yeast (Saccharomyces cerevisiae) and human cells to be captured and, excitingly, these structures not only allow pre-rRNAs to be observed before and after cleavage events, but also enable ribonucleases to be visualized on their target RNAs. These structural views of pre-rRNA processing in action allow a new layer of understanding of rRNA maturation and how it is coordinated with other aspects of ribosome assembly. They illuminate mechanisms of target recognition by the diverse ribonucleases involved and reveal how the cleavage/processing activities of these enzymes are regulated. In this review, we discuss the new insights into pre-rRNA processing gained by structural analyses and the growing understanding of the mechanisms of ribonuclease regulation. This article is categorized under: Translation > Ribosome Biogenesis RNA Processing > rRNA Processing.
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Affiliation(s)
- Claudia Schneider
- Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK
| | - Katherine E Bohnsack
- Department of Molecular Biology, University Medical Center Göttingen, Göttingen, Germany
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Dörner K, Ruggeri C, Zemp I, Kutay U. Ribosome biogenesis factors-from names to functions. EMBO J 2023; 42:e112699. [PMID: 36762427 PMCID: PMC10068337 DOI: 10.15252/embj.2022112699] [Citation(s) in RCA: 79] [Impact Index Per Article: 39.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2022] [Revised: 12/13/2022] [Accepted: 01/19/2023] [Indexed: 02/11/2023] Open
Abstract
The assembly of ribosomal subunits is a highly orchestrated process that involves a huge cohort of accessory factors. Most eukaryotic ribosome biogenesis factors were first identified by genetic screens and proteomic approaches of pre-ribosomal particles in Saccharomyces cerevisiae. Later, research on human ribosome synthesis not only demonstrated that the requirement for many of these factors is conserved in evolution, but also revealed the involvement of additional players, reflecting a more complex assembly pathway in mammalian cells. Yet, it remained a challenge for the field to assign a function to many of the identified factors and to reveal their molecular mode of action. Over the past decade, structural, biochemical, and cellular studies have largely filled this gap in knowledge and led to a detailed understanding of the molecular role that many of the players have during the stepwise process of ribosome maturation. Such detailed knowledge of the function of ribosome biogenesis factors will be key to further understand and better treat diseases linked to disturbed ribosome assembly, including ribosomopathies, as well as different types of cancer.
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Affiliation(s)
- Kerstin Dörner
- Department of Biology, Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.,Molecular Life Sciences Ph.D. Program, Zurich, Switzerland
| | - Chiara Ruggeri
- Department of Biology, Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.,RNA Biology Ph.D. Program, Zurich, Switzerland
| | - Ivo Zemp
- Department of Biology, Institute of Biochemistry, ETH Zurich, Zurich, Switzerland
| | - Ulrike Kutay
- Department of Biology, Institute of Biochemistry, ETH Zurich, Zurich, Switzerland
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10
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Celik B, Cicek K, Leal AF, Tomatsu S. Regulation of Molecular Targets in Osteosarcoma Treatment. Int J Mol Sci 2022; 23:12583. [PMID: 36293439 PMCID: PMC9604206 DOI: 10.3390/ijms232012583] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2022] [Revised: 10/15/2022] [Accepted: 10/17/2022] [Indexed: 11/17/2022] Open
Abstract
The most prevalent malignant bone tumor, osteosarcoma, affects the growth plates of long bones in adolescents and young adults. Standard chemotherapeutic methods showed poor response rates in patients with recurrent and metastatic phases. Therefore, it is critical to develop novel and efficient targeted therapies to address relapse cases. In this regard, RNA interference technologies are encouraging options in cancer treatment, in which small interfering RNAs regulate the gene expression following RNA interference pathways. The determination of target tissue is as important as the selection of tissue-specific promoters. Moreover, small interfering RNAs should be delivered effectively into the cytoplasm. Lentiviral vectors could encapsulate and deliver the desired gene into the cell and integrate it into the genome, providing long-term regulation of targeted genes. Silencing overexpressed genes promote the tumor cells to lose invasiveness, prevents their proliferation, and triggers their apoptosis. The uniqueness of cancer cells among patients requires novel therapeutic methods that treat patients based on their unique mutations. Several studies showed the effectiveness of different approaches such as microRNA, drug- or chemotherapy-related methods in treating the disease; however, identifying various targets was challenging to understanding disease progression. In this regard, the patient-specific abnormal gene might be targeted using genomics and molecular advancements such as RNA interference approaches. Here, we review potential therapeutic targets for the RNA interference approach, which is applicable as a therapeutic option for osteosarcoma patients, and we point out how the small interfering RNA method becomes a promising approach for the unmet challenge.
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Affiliation(s)
- Betul Celik
- Department of Biological Science, University of Delaware, Newark, DE 19716, USA
- Nemours/Alfred I. DuPont Hospital for Children, Wilmington, DE 19803, USA
| | - Kader Cicek
- Department of Biological Science, University of Delaware, Newark, DE 19716, USA
- Nemours/Alfred I. DuPont Hospital for Children, Wilmington, DE 19803, USA
| | - Andrés Felipe Leal
- Nemours/Alfred I. DuPont Hospital for Children, Wilmington, DE 19803, USA
| | - Shunji Tomatsu
- Nemours/Alfred I. DuPont Hospital for Children, Wilmington, DE 19803, USA
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11
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Shi Z, Zhou X, Bao M, Jia R, Chu Y, Lin Y. miRNA-612 suppresses ovarian cancer cell tumorigenicity by downregulating NOB1. Am J Transl Res 2022; 14:3904-3914. [PMID: 35836846 PMCID: PMC9274555] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2022] [Accepted: 03/31/2022] [Indexed: 06/15/2023]
Abstract
MicroRNAs (miRNAs) play crucial roles in cancer progression. Our previous study demonstrated that NIN1/RPN12 binding protein 1 homolog (NOB1) was a functional regulator in the progression of ovarian cancer (OC). However, the role of miRNA-612 (miR-612) in OC has not been elucidated. In this study, we aimed to investigate the regulatory mechanism of NOB1 targeting miRNA, miR-612, in OC tumorigenicity. The miR-612 expression was down-regulated in OC patient tissues and four OC cell lines (Caov3, A2780, SKOV3 and OVCAR3). The miR-612 level was negatively correlated with NOB1 expression, and dual-luciferase reporter assay indicated that miR-612 suppressed NOB1 expression by targeting the 3'UTR of NOB1 transcript. Up-regulation of miR-612 mediated by lentiviral transduction suppressed cell proliferation, colony formation, migration, invasion, and induced apoptosis in OC cell lines. In addition, miR-612 overexpression inhibited tumor growth of OC in vivo by sequestering NOB1 expression. In conclusion, our results suggested that miR-612 directly targeted NOB1 to suppress OC progression. Therefore, the miR-612-NOB1 axis could serve as therapeutic targets for OC.
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Affiliation(s)
- Zhikun Shi
- Department of Gynecology and Obstetrics, The Second Hospital of Jilin University Changchun 130041, Jilin, China
| | - Xu Zhou
- Department of Gynecology and Obstetrics, The Second Hospital of Jilin University Changchun 130041, Jilin, China
| | - Meijing Bao
- Department of Gynecology and Obstetrics, The Second Hospital of Jilin University Changchun 130041, Jilin, China
| | - Rongxia Jia
- Department of Gynecology and Obstetrics, The Second Hospital of Jilin University Changchun 130041, Jilin, China
| | - Yuqing Chu
- Department of Gynecology and Obstetrics, The Second Hospital of Jilin University Changchun 130041, Jilin, China
| | - Yang Lin
- Department of Gynecology and Obstetrics, The Second Hospital of Jilin University Changchun 130041, Jilin, China
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12
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Hurtig JE, van Hoof A. Yeast Dxo1 is required for 25S rRNA maturation and acts as a transcriptome-wide distributive exonuclease. RNA (NEW YORK, N.Y.) 2022; 28:657-667. [PMID: 35140172 PMCID: PMC9014881 DOI: 10.1261/rna.078952.121] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/13/2021] [Accepted: 01/24/2022] [Indexed: 05/03/2023]
Abstract
The Dxo1/Rai1/DXO family of decapping and exonuclease enzymes can catalyze the in vitro removal of chemically diverse 5' ends from RNA. Specifically, these enzymes act poorly on RNAs with a canonical 7mGpppN cap, but instead prefer RNAs with a triphosphate, monophosphate, hydroxyl, or nonconventional cap. In each case, these enzymes generate an RNA with a 5' monophosphate, which is then thought to be further degraded by Rat1/Xrn1 5' exoribonucleases. For most Dxo1/Rai1/DXO family members, it is not known which of these activities is most important in vivo. Here we describe the in vivo function of the poorly characterized cytoplasmic family member, yeast Dxo1. Using RNA-seq of 5' monophosphate ends, we show that Dxo1 can act as a distributive exonuclease, removing a few nucleotides from endonuclease or decapping products. We also show that Dxo1 is required for the final 5' end processing of 25S rRNA, and that this is the primary role of Dxo1. While Dxo1/Rai1/DXO members were expected to act upstream of Rat1/Xrn1, this order is reversed in 25S rRNA processing, with Dxo1 acting downstream from Rat1. Such a hand-off from a processive to a distributive exonuclease may be a general phenomenon in the precise maturation of RNA ends.
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Affiliation(s)
- Jennifer E Hurtig
- Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas 77030, USA
| | - Ambro van Hoof
- Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas 77030, USA
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13
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YbeY, éminence grise of ribosome biogenesis. Biochem Soc Trans 2021; 49:727-745. [PMID: 33929506 DOI: 10.1042/bst20200669] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2021] [Revised: 04/12/2021] [Accepted: 04/14/2021] [Indexed: 12/30/2022]
Abstract
YbeY is an ultraconserved small protein belonging to the unique heritage shared by most existing bacteria and eukaryotic organelles of bacterial origin, mitochondria and chloroplasts. Studied in more than a dozen of evolutionarily distant species, YbeY is invariably critical for cellular physiology. However, the exact mechanisms by which it exerts such penetrating influence are not completely understood. In this review, we attempt a transversal analysis of the current knowledge about YbeY, based on genetic, structural, and biochemical data from a wide variety of models. We propose that YbeY, in association with the ribosomal protein uS11 and the assembly GTPase Era, plays a critical role in the biogenesis of the small ribosomal subunit, and more specifically its platform region, in diverse genetic systems of bacterial type.
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14
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Ke W, Lu Z, Zhao X. NOB1: A Potential Biomarker or Target in Cancer. Curr Drug Targets 2020; 20:1081-1089. [PMID: 30854959 DOI: 10.2174/1389450120666190308145346] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2018] [Revised: 02/06/2019] [Accepted: 03/05/2019] [Indexed: 12/18/2022]
Abstract
Human NIN1/RPN12 binding protein 1 homolog (NOB1), an RNA binding protein, is expressed ubiquitously in normal tissues such as the lung, liver, and spleen. Its core physiological function is to regulate protease activities and participate in maintaining RNA metabolism and stability. NOB1 is overexpressed in a variety of cancers, including pancreatic cancer, non-small cell lung cancer, ovarian cancer, prostate carcinoma, osteosarcoma, papillary thyroid carcinoma, colorectal cancer, and glioma. Although existing data indicate that NOB1 overexpression is associated with cancer growth, invasion, and poor prognosis, the molecular mechanisms behind these effects and its exact roles remain unclear. Several studies have confirmed that NOB1 is clinically relevant in different cancers, and further research at the molecular level will help evaluate the role of NOB1 in tumors. NOB1 has become an attractive target in anticancer therapy because it is overexpressed in many cancers and mediates different stages of tumor development. Elucidating the role of NOB1 in different signaling pathways as a potential cancer treatment will provide new ideas for existing cancer treatment methods. This review summarizes the research progress made into NOB1 in cancer in the past decade; this information provides valuable clues and theoretical guidance for future anticancer therapy by targeting NOB1.
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Affiliation(s)
- Weiwei Ke
- Department of Radiology, Shengjing Hospital of China Medical University, Shenyang 110004, LN, China
| | - Zaiming Lu
- Department of Radiology, Shengjing Hospital of China Medical University, Shenyang 110004, LN, China
| | - Xiangxuan Zhao
- Department of Radiology, Shengjing Hospital of China Medical University, Shenyang 110004, LN, China
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15
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Martín-Villanueva S, Fernández-Fernández J, Rodríguez-Galán O, Fernández-Boraita J, Villalobo E, de La Cruz J. Role of the 40S beak ribosomal protein eS12 in ribosome biogenesis and function in Saccharomyces cerevisiae. RNA Biol 2020; 17:1261-1276. [PMID: 32408794 DOI: 10.1080/15476286.2020.1767951] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022] Open
Abstract
In eukaryotes, the beak structure of 40S subunits is formed by the protrusion of the 18S rRNA helix 33 and three ribosomal proteins: eS10, eS12 and eS31. The exact role of these proteins in ribosome biogenesis is not well understood. While eS10 is an essential protein encoded by two paralogous genes in Saccharomyces cerevisiae, eS12 and eS31 are not essential proteins encoded by the single-copy genes RPS12 and UBI3, respectively. Here, we have analysed the contribution of yeast eS12 to ribosome biogenesis and compared it with that of eS31. Polysome analysis reveals that deletion of either RPS12 or UBI3 results in equivalent 40S deficits. Analysis of pre-rRNA processing indicates that eS12, akin to eS31, is required for efficient processing of 20S pre-rRNA to mature 18S rRNA. Moreover, we show that the 20S pre-rRNA accumulates within cytoplasmic pre-40S particles, as deduced from FISH experiments and the lack of nuclear retention of 40S subunit reporter proteins, in rps12∆ and ubi3∆ cells. However, these particles containing 20S pre-rRNA are not efficiently incorporated into polyribosomes. We also provide evidence for a genetic interaction between eS12 or eS31 and the late-acting 40S assembly factors Enp1 and Ltv1, which appears not to be linked to the dynamics of their association with or release from pre-40S particles in the absence of either eS12 or eS31. Finally, we show that eS12- and eS31-deficient ribosomes exhibit increased levels of translational misreading. Altogether, our data highlight distinct important roles of the beak region during ribosome assembly and function.
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Affiliation(s)
- Sara Martín-Villanueva
- Departamento de Genética, Facultad de Biología, Universidad de Sevilla , Seville, Spain.,Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla , Seville, Spain
| | - José Fernández-Fernández
- Departamento de Genética, Facultad de Biología, Universidad de Sevilla , Seville, Spain.,Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla , Seville, Spain
| | - Olga Rodríguez-Galán
- Departamento de Genética, Facultad de Biología, Universidad de Sevilla , Seville, Spain.,Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla , Seville, Spain
| | - Julia Fernández-Boraita
- Departamento de Genética, Facultad de Biología, Universidad de Sevilla , Seville, Spain.,Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla , Seville, Spain
| | - Eduardo Villalobo
- Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla , Seville, Spain.,Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla , Seville, Spain
| | - Jesús de La Cruz
- Departamento de Genética, Facultad de Biología, Universidad de Sevilla , Seville, Spain.,Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla , Seville, Spain
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16
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Jurėnas D, Van Melderen L. The Variety in the Common Theme of Translation Inhibition by Type II Toxin-Antitoxin Systems. Front Genet 2020; 11:262. [PMID: 32362907 PMCID: PMC7180214 DOI: 10.3389/fgene.2020.00262] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2019] [Accepted: 03/05/2020] [Indexed: 12/12/2022] Open
Abstract
Type II Toxin-antitoxin (TA) modules are bacterial operons that encode a toxic protein and its antidote, which form a self-regulating genetic system. Antitoxins put a halter on toxins in many ways that distinguish different types of TA modules. In type II TA modules, toxin and antitoxin are proteins that form a complex which physically sequesters the toxin, thereby preventing its toxic activity. Type II toxins inhibit various cellular processes, however, the translation process appears to be their favorite target and nearly every step of this complex process is inhibited by type II toxins. The structural features, enzymatic activities and target specificities of the different toxin families are discussed. Finally, this review emphasizes that the structural folds presented by these toxins are not restricted to type II TA toxins or to one particular cellular target, and discusses why so many of them evolved to target translation as well as the recent developments regarding the role(s) of these systems in bacterial physiology and evolution.
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Affiliation(s)
- Dukas Jurėnas
- Laboratoire d’Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie de la Méditerranée, CNRS, Aix-Marseille Université, Marseille, France
| | - Laurence Van Melderen
- Cellular and Molecular Microbiology, Faculté des Sciences, Université libre de Bruxelles, Gosselies, Belgium
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17
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Ren Z, Yao L, Liu J, Qi Z, Li J. Silencing NOB1 Can Affect Cell Proliferation and Apoptosis Via the C-Jun N-Terminal Kinase Pathway in Colorectal Cancer. J INVEST SURG 2020; 34:819-825. [PMID: 31906747 DOI: 10.1080/08941939.2019.1697401] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
OBJECTIVES To investigate the bio-functions and the molecular mechanisms of NIN1/proteasome 26S subunit non-ATPase 8 binding protein 1 homolog (NOB1) in colorectal cancer cells. METHODS NOB1 expression was silenced using si-RNA in SW480 and LoVo cells. The transfection efficiency was measured by western blotting and RT-qPCR. Subsequently, the proliferation of SW480 and LoVo cells was determined using both MTT assay and colony-formation assay. Apoptosis and cell cycle analysis were determined using flow cytometry. RESULTS Compared with the normal control (NC) and scramble cells, si-NOB1 could significantly attenuate the proliferation, colony-formation ability and cell percentage of S stage (p < 0.05). Additionally, at the phosphorylation level, si-NOB1 could notably increase the expression of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38. CONCLUSIONS Inhibition of NOB1 expression suppressed the proliferation, and promoted the apoptosis through regulation of the JNK signaling pathway.
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Affiliation(s)
- Zhong Ren
- Endoscopy Center, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Liqing Yao
- Endoscopy Center, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Jingzheng Liu
- Endoscopy Center, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Zhipeng Qi
- Endoscopy Center, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Jian Li
- Endoscopy Center, Zhongshan Hospital, Fudan University, Shanghai, China
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18
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Parker MD, Collins JC, Korona B, Ghalei H, Karbstein K. A kinase-dependent checkpoint prevents escape of immature ribosomes into the translating pool. PLoS Biol 2019; 17:e3000329. [PMID: 31834877 PMCID: PMC6934326 DOI: 10.1371/journal.pbio.3000329] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2019] [Revised: 12/27/2019] [Accepted: 11/29/2019] [Indexed: 12/14/2022] Open
Abstract
Premature release of nascent ribosomes into the translating pool must be prevented because these do not support viability and may be prone to mistakes. Here, we show that the kinase Rio1, the nuclease Nob1, and its binding partner Pno1 cooperate to establish a checkpoint that prevents the escape of immature ribosomes into polysomes. Nob1 blocks mRNA recruitment, and rRNA cleavage is required for its dissociation from nascent 40S subunits, thereby setting up a checkpoint for maturation. Rio1 releases Nob1 and Pno1 from pre-40S ribosomes to discharge nascent 40S into the translating pool. Weak-binding Nob1 and Pno1 mutants can bypass the requirement for Rio1, and Pno1 mutants rescue cell viability. In these strains, immature ribosomes escape into the translating pool, where they cause fidelity defects and perturb protein homeostasis. Thus, the Rio1-Nob1-Pno1 network establishes a checkpoint that safeguards against the release of immature ribosomes into the translating pool.
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Affiliation(s)
- Melissa D. Parker
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, Jupiter, Florida, United States of America
| | - Jason C. Collins
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, Jupiter, Florida, United States of America
| | - Boguslawa Korona
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, Jupiter, Florida, United States of America
| | - Homa Ghalei
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, Jupiter, Florida, United States of America
| | - Katrin Karbstein
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, Jupiter, Florida, United States of America
- HHMI Faculty Scholar, Chevy Chase, Maryland, United States of America
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19
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Tsr4 Is a Cytoplasmic Chaperone for the Ribosomal Protein Rps2 in Saccharomyces cerevisiae. Mol Cell Biol 2019; 39:MCB.00094-19. [PMID: 31182640 DOI: 10.1128/mcb.00094-19] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2019] [Accepted: 06/03/2019] [Indexed: 01/31/2023] Open
Abstract
Eukaryotic ribosome biogenesis requires the action of approximately 200 trans-acting factors and the incorporation of 79 ribosomal proteins (RPs). The delivery of RPs to preribosomes is a major challenge for the cell because RPs are often highly basic and contain intrinsically disordered regions prone to nonspecific interactions and aggregation. To counteract this, eukaryotes developed dedicated chaperones for certain RPs that promote their solubility and expression, often by binding eukaryote-specific extensions of the RPs. Rps2 (uS5) is a universally conserved RP that assembles into nuclear pre-40S subunits. However, a chaperone for Rps2 had not been identified. Our laboratory previously characterized Tsr4 as a 40S biogenesis factor of unknown function. Here, we report that Tsr4 cotranslationally associates with Rps2. Rps2 harbors a eukaryote-specific N-terminal extension that is critical for its interaction with Tsr4. Moreover, Tsr4 perturbation resulted in decreased Rps2 levels and phenocopied Rps2 depletion. Despite Rps2 joining nuclear pre-40S particles, Tsr4 appears to be restricted to the cytoplasm. Thus, we conclude that Tsr4 is a cytoplasmic chaperone dedicated to Rps2.
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20
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Ke W, Lu Z, Zhao X. NOB1: A Potential Biomarker or Target in Cancer. Curr Drug Targets 2019; 20:1081-1089. [DOI: doi10.2174/1389450120666190308145346] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2018] [Revised: 02/06/2019] [Accepted: 03/05/2019] [Indexed: 09/01/2023]
Abstract
Human NIN1/RPN12 binding protein 1 homolog (NOB1), an RNA binding protein, is expressed ubiquitously in normal tissues such as the lung, liver, and spleen. Its core physiological function is to regulate protease activities and participate in maintaining RNA metabolism and stability. NOB1 is overexpressed in a variety of cancers, including pancreatic cancer, non-small cell lung cancer, ovarian cancer, prostate carcinoma, osteosarcoma, papillary thyroid carcinoma, colorectal cancer, and glioma. Although existing data indicate that NOB1 overexpression is associated with cancer growth, invasion, and poor prognosis, the molecular mechanisms behind these effects and its exact roles remain unclear. Several studies have confirmed that NOB1 is clinically relevant in different cancers, and further research at the molecular level will help evaluate the role of NOB1 in tumors. NOB1 has become an attractive target in anticancer therapy because it is overexpressed in many cancers and mediates different stages of tumor development. Elucidating the role of NOB1 in different signaling pathways as a potential cancer treatment will provide new ideas for existing cancer treatment methods. This review summarizes the research progress made into NOB1 in cancer in the past decade; this information provides valuable clues and theoretical guidance for future anticancer therapy by targeting NOB1.
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Affiliation(s)
- Weiwei Ke
- Department of Radiology, Shengjing Hospital of China Medical University, Shenyang 110004, LN, China
| | - Zaiming Lu
- Department of Radiology, Shengjing Hospital of China Medical University, Shenyang 110004, LN, China
| | - Xiangxuan Zhao
- Department of Radiology, Shengjing Hospital of China Medical University, Shenyang 110004, LN, China
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21
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Clouet-d'Orval B, Batista M, Bouvier M, Quentin Y, Fichant G, Marchfelder A, Maier LK. Insights into RNA-processing pathways and associated RNA-degrading enzymes in Archaea. FEMS Microbiol Rev 2018; 42:579-613. [PMID: 29684129 DOI: 10.1093/femsre/fuy016] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2018] [Accepted: 04/17/2018] [Indexed: 12/20/2022] Open
Abstract
RNA-processing pathways are at the centre of regulation of gene expression. All RNA transcripts undergo multiple maturation steps in addition to covalent chemical modifications to become functional in the cell. This includes destroying unnecessary or defective cellular RNAs. In Archaea, information on mechanisms by which RNA species reach their mature forms and associated RNA-modifying enzymes are still fragmentary. To date, most archaeal actors and pathways have been proposed in light of information gathered from Bacteria and Eukarya. In this context, this review provides a state of the art overview of archaeal endoribonucleases and exoribonucleases that cleave and trim RNA species and also of the key small archaeal proteins that bind RNAs. Furthermore, synthetic up-to-date views of processing and biogenesis pathways of archaeal transfer and ribosomal RNAs as well as of maturation of stable small non-coding RNAs such as CRISPR RNAs, small C/D and H/ACA box guide RNAs, and other emerging classes of small RNAs are described. Finally, prospective post-transcriptional mechanisms to control archaeal messenger RNA quality and quantity are discussed.
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Affiliation(s)
- Béatrice Clouet-d'Orval
- Laboratoire de Microbiologie et de Génétique Moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, 31062 Toulouse, France
| | - Manon Batista
- Laboratoire de Microbiologie et de Génétique Moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, 31062 Toulouse, France
| | - Marie Bouvier
- Laboratoire de Microbiologie et de Génétique Moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, 31062 Toulouse, France
| | - Yves Quentin
- Laboratoire de Microbiologie et de Génétique Moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, 31062 Toulouse, France
| | - Gwennaele Fichant
- Laboratoire de Microbiologie et de Génétique Moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, 31062 Toulouse, France
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22
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Cerezo E, Plisson-Chastang C, Henras AK, Lebaron S, Gleizes PE, O'Donohue MF, Romeo Y, Henry Y. Maturation of pre-40S particles in yeast and humans. WILEY INTERDISCIPLINARY REVIEWS-RNA 2018; 10:e1516. [PMID: 30406965 DOI: 10.1002/wrna.1516] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/15/2018] [Revised: 09/02/2018] [Accepted: 10/01/2018] [Indexed: 12/22/2022]
Abstract
The synthesis of ribosomal subunits in eukaryotes requires the interplay of numerous maturation and assembly factors (AFs) that intervene in the insertion of ribosomal proteins within pre-ribosomal particles, the ribosomal subunit precursors, as well as in pre-ribosomal RNA (rRNA) processing and folding. Here, we review the intricate nuclear and cytoplasmic maturation steps of pre-40S particles, the precursors to the small ribosomal subunits, in both yeast and human cells, with particular emphasis on the timing and mechanisms of AF association with and dissociation from pre-40S particles and the roles of these AFs in the maturation process. We highlight the particularly complex pre-rRNA processing pathway in human cells, compared to yeast, to generate the mature 18S rRNA. We discuss the information gained from the recently published cryo-electron microscopy atomic models of yeast and human pre-40S particles, as well as the checkpoint/quality control systems that seem to operate to probe functional sites within yeast cytoplasmic pre-40S particles. This article is categorized under: RNA Processing > rRNA Processing Translation > Ribosome Biogenesis.
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Affiliation(s)
- Emilie Cerezo
- Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France
| | - Célia Plisson-Chastang
- Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France
| | - Anthony K Henras
- Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France
| | - Simon Lebaron
- Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France
| | - Pierre-Emmanuel Gleizes
- Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France
| | - Marie-Françoise O'Donohue
- Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France
| | - Yves Romeo
- Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France
| | - Yves Henry
- Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France
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23
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Raoelijaona F, Thore S, Fribourg S. Domain definition and interaction mapping for the endonuclease complex hNob1/hPno1. RNA Biol 2018; 15:1174-1180. [PMID: 30176151 DOI: 10.1080/15476286.2018.1517013] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/28/2022] Open
Abstract
Ribosome biogenesis requires a variety of trans-acting factors in order to produce functional ribosomal subunits. In human cells, the complex formed by the proteins hNob1 and hPno1 is crucial to the site 3 cleavage occurring at the 3'-end of 18S pre-rRNA. However, the properties and activity of this complex are still poorly understood. We present here a detailed characterization of hNob1 organization and its interaction with hPno1. We redefine the boundaries of the endonuclease PIN domain present in hNob1 and we further delineate the precise interacting modules required for complex formation in hNob1 and hPno1. Altogether, our data contributes to a better understanding of the complex biology required during the site 3 cleavage step in ribosome biogenesis.
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Affiliation(s)
| | - Stéphane Thore
- a INSERM U1212, CNRS UMR5320 , Université de Bordeaux , Bordeaux , France
| | - Sébastien Fribourg
- a INSERM U1212, CNRS UMR5320 , Université de Bordeaux , Bordeaux , France
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24
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Chaker-Margot M. Assembly of the small ribosomal subunit in yeast: mechanism and regulation. RNA (NEW YORK, N.Y.) 2018; 24:881-891. [PMID: 29712726 PMCID: PMC6004059 DOI: 10.1261/rna.066985.118] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/05/2023]
Abstract
The eukaryotic ribosome is made of four intricately folded ribosomal RNAs and 79 proteins. During rapid growth, yeast cells produce an incredible 2000 ribosomes every minute. Ribosome assembly involves more than 200 trans-acting factors, intervening from the transcription of the preribosomal RNA in the nucleolus to late maturation events in the cytoplasm. The biogenesis of the small ribosomal subunit, or 40S, is especially intricate, requiring more than four times the mass of the small subunit in assembly factors for its full maturation. Recent studies have provided new insights into the complex assembly of the 40S subunit. These data from cryo-electron microscopy, X-ray crystallography, and other biochemical and molecular biology methods, have elucidated the role of many factors required in small subunit maturation. Mechanisms of the regulation of ribosome assembly have also emerged from this body of work. This review aims to integrate these new results into an updated view of small subunit biogenesis and its regulation, in yeast, from transcription to the formation of the mature small subunit.
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Affiliation(s)
- Malik Chaker-Margot
- The Rockefeller University, New York, New York 10065, USA
- Tri-Institutional Program in Chemical Biology, New York, New York 10065, USA
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25
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Substrate specificity of human MCPIP1 endoribonuclease. Sci Rep 2018; 8:7381. [PMID: 29743536 PMCID: PMC5943514 DOI: 10.1038/s41598-018-25765-2] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2017] [Accepted: 04/27/2018] [Indexed: 12/20/2022] Open
Abstract
MCPIP1, also known as Regnase-1, is a ribonuclease crucial for regulation of stability of transcripts related to inflammatory processes. Here, we report that MCPIP1 acts as an endonuclease by degrading several stem-loop RNA structures and single-stranded RNAs. Our studies revealed cleavage sites present in the stem-loops derived from the 3′ untranslated region of the interleukin-6 transcript. Furthermore, MCPIP1 induced endonuclease cleavage at the loop motif of stem-loop structures. Additionally, we observed that MCPIP1 could cleave single-stranded RNA fragments. However, RNA substrates shorter than 6 nucleotides were not further affected by MCPIP1 nucleolytic activity. In this study, we also determined the dissociation constants of full-length MCPIP1D141N and its ribonuclease domain PIN D141N with twelve oligonucleotides substrates. The equilibrium binding constants (Kd) for MCPIP1D141N and the RNA targets were approximately 10 nM. Interestingly, we observed that the presence of a zinc finger in the PIN domain increases the affinity of this protein fragment to 25-nucleotide-long stem-loop RNA but not to shorter ones. Furthermore, size exclusion chromatography of the MCPIP1 and PIN proteins suggested that MCPIP1 undergoes homooligomerization during interaction with RNA substrates. Our results provide insight into the mechanism of MCPIP1 substrate recognition and its affinity towards various oligonucleotides.
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26
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An W, Du Y, Ye K. Structural and functional analysis of Utp24, an endonuclease for processing 18S ribosomal RNA. PLoS One 2018; 13:e0195723. [PMID: 29641590 PMCID: PMC5895043 DOI: 10.1371/journal.pone.0195723] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2018] [Accepted: 03/28/2018] [Indexed: 01/05/2023] Open
Abstract
The precursor ribosomal RNA is processed by multiple steps of nucleolytic cleavage to generate mature rRNAs. Utp24 is a PIN domain endonuclease in the early 90S precursor of small ribosomal subunit and is proposed to cleave at sites A1 and A2 of pre-rRNA. Here we determine the crystal structure of Utp24 from Schizosaccharomyces pombe at 2.1 angstrom resolution. Utp24 structurally resembles the ribosome assembly factor Utp23 and both contain a Zn-finger motif. Functional analysis in Saccharomyces cerevisiae shows that depletion of Utp24 disturbs the assembly of 90S and abolishes cleavage at sites A0, A1 and A2. The 90S assembled with inactivated Utp24 is arrested at a post-A0-cleavage state and contains enriched nuclear exosome for degradation of 5' ETS. Despite of high sequence conservation, Utp24 from other organisms is unable to form an active 90S in S. cerevisiae, suggesting that Utp24 needs to be precisely positioned in 90S. Our study provides biochemical and structural insight into the role of Utp24 in 90S assembly and activity.
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Affiliation(s)
- Weidong An
- College of Biological Sciences, China Agricultural University, Beijing, China
- National Institute of Biological Sciences, Beijing, China
- Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
| | - Yifei Du
- Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Keqiong Ye
- Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
- * E-mail:
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27
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Scaiola A, Peña C, Weisser M, Böhringer D, Leibundgut M, Klingauf-Nerurkar P, Gerhardy S, Panse VG, Ban N. Structure of a eukaryotic cytoplasmic pre-40S ribosomal subunit. EMBO J 2018; 37:embj.201798499. [PMID: 29459436 DOI: 10.15252/embj.201798499] [Citation(s) in RCA: 72] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2017] [Revised: 12/18/2017] [Accepted: 01/10/2018] [Indexed: 11/09/2022] Open
Abstract
Final maturation of eukaryotic ribosomes occurs in the cytoplasm and requires the sequential removal of associated assembly factors and processing of the immature 20S pre-RNA Using cryo-electron microscopy (cryo-EM), we have determined the structure of a yeast cytoplasmic pre-40S particle in complex with Enp1, Ltv1, Rio2, Tsr1, and Pno1 assembly factors poised to initiate final maturation. The structure reveals that the pre-rRNA adopts a highly distorted conformation of its 3' major and 3' minor domains stabilized by the binding of the assembly factors. This observation is consistent with a mechanism that involves concerted release of the assembly factors orchestrated by the folding of the rRNA in the head of the pre-40S subunit during the final stages of maturation. Our results provide a structural framework for the coordination of the final maturation events that drive a pre-40S particle toward the mature form capable of engaging in translation.
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Affiliation(s)
- Alain Scaiola
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland
| | - Cohue Peña
- Department of Biology, Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.,Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland
| | - Melanie Weisser
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland
| | - Daniel Böhringer
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland
| | - Marc Leibundgut
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland
| | - Purnima Klingauf-Nerurkar
- Department of Biology, Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.,Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland
| | - Stefan Gerhardy
- Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland
| | - Vikram Govind Panse
- Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland
| | - Nenad Ban
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland
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28
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Luo L, Wang Y, Yin Y, Ge J, Lu X. Effects of NOB1 on the pathogenesis of osteosarcoma and its expression on the chemosensitivity to cisplatin. Oncol Lett 2018; 15:3548-3551. [PMID: 29467874 PMCID: PMC5796338 DOI: 10.3892/ol.2018.7730] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2017] [Accepted: 11/23/2017] [Indexed: 12/19/2022] Open
Abstract
The effects of NIN1/RPN12 binding protein 1 homolog (NOB1) on the pathogenesis of osteosarcoma and its expression on the chemosensitivity to cisplatin were investigated. Seventy-four patients with osteosarcoma who received surgical resection in The Affiliated Hospital of Southwest Medical University (Sichuan, China) from September 2013 to September 2016 were enrolled in this study. The expression of NOB1 in cancer and cancer-adjacent tissues of patients was detected by reverse transcription-polymerase chain reaction, and the relationship between NOB1 expression and the pathogenesis of osteosarcoma was analyzed. The expression of NOB1 in osteosarcoma MG-63 cells was interfered with using small interfering ribonucleic acid (siRNA). Western blotting was used to detect the transfection efficiency and changes in apoptosis indicators. Cell Counting Kit-8 (CCK-8) assay was used to examine changes in the sensitivity of cells to cisplatin. The effect of NOB1 knockout on cell apoptosis was examined by flow cytometry. In patients with osteosarcoma, the level of NOB1 mRNA in cancer tissues was significantly higher than that in cancer-adjacent tissues (p<0.05), and the expression of NOB1 was correlated with Ennecking staging and tumor size (p<0.05). The expression level of the apoptotic indicator caspase-3 was activated after siRNA interfered with NOB1 expression, thus reducing the expression level of anti-apoptotic indicator B-cell lymphoma 2. CCK-8 results showed that the downregulation of NOB1 increased the sensitivity of MG-63 cells to cisplatin (p<0.05). In addition, flow cytometry showed that the downregulation of NOB1 significantly promoted the apoptosis of MG-63 cells. NOB1 is significantly upregulated in patients with osteosarcoma, thus reducing the curative effect of cisplatin chemotherapy, which indicates that the prognosis is poor.
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Affiliation(s)
- Leiming Luo
- Department of Bone and Joint Surgery, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, P.R. China
| | - Yuanhui Wang
- Department of Bone and Joint Surgery, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, P.R. China
| | - Yiran Yin
- Department of Bone and Joint Surgery, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, P.R. China
| | - Jianhua Ge
- Department of Bone and Joint Surgery, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, P.R. China
| | - Xiaobo Lu
- Department of Bone and Joint Surgery, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, P.R. China
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29
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Montellese C, Montel-Lehry N, Henras AK, Kutay U, Gleizes PE, O'Donohue MF. Poly(A)-specific ribonuclease is a nuclear ribosome biogenesis factor involved in human 18S rRNA maturation. Nucleic Acids Res 2017; 45:6822-6836. [PMID: 28402503 PMCID: PMC5499762 DOI: 10.1093/nar/gkx253] [Citation(s) in RCA: 42] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2016] [Accepted: 04/03/2017] [Indexed: 01/28/2023] Open
Abstract
The poly-A specific ribonuclease (PARN), initially characterized for its role in mRNA catabolism, supports the processing of different types of non-coding RNAs including telomerase RNA. Mutations in PARN are linked to dyskeratosis congenita and pulmonary fibrosis. Here, we show that PARN is part of the enzymatic machinery that matures the human 18S ribosomal RNA (rRNA). Consistent with its nucleolar steady-state localization, PARN is required for 40S ribosomal subunit production and co-purifies with 40S subunit precursors. Depletion of PARN or expression of a catalytically-compromised PARN mutant results in accumulation of 3΄ extended 18S rRNA precursors. Analysis of these processing intermediates reveals a defect in 3΄ to 5΄ trimming of the internal transcribed spacer 1 (ITS1) region, subsequent to endonucleolytic cleavage at site E. Consistent with a function of PARN in exonucleolytic trimming of 18S-E pre-rRNA, recombinant PARN can process the corresponding ITS1 RNA fragment in vitro. Trimming of 18S-E pre-rRNA by PARN occurs in the nucleus, upstream of the final endonucleolytic cleavage by the endonuclease NOB1 in the cytoplasm. These results identify PARN as a new component of the ribosome biogenesis machinery in human cells. Defects in ribosome biogenesis could therefore underlie the pathologies linked to mutations in PARN.
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Affiliation(s)
| | - Nathalie Montel-Lehry
- Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, 31000 Toulouse, France
| | - Anthony K Henras
- Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, 31000 Toulouse, France
| | - Ulrike Kutay
- Institut für Biochemie, ETH Zurich, Zurich CH-8093, Switzerland
| | - Pierre-Emmanuel Gleizes
- Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, 31000 Toulouse, France
| | - Marie-Françoise O'Donohue
- Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, 31000 Toulouse, France
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30
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Barandun J, Chaker-Margot M, Hunziker M, Molloy KR, Chait BT, Klinge S. The complete structure of the small-subunit processome. Nat Struct Mol Biol 2017; 24:944-953. [PMID: 28945246 DOI: 10.1038/nsmb.3472] [Citation(s) in RCA: 104] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2017] [Accepted: 08/25/2017] [Indexed: 01/24/2023]
Abstract
The small-subunit processome represents the earliest stable precursor of the eukaryotic small ribosomal subunit. Here we present the cryo-EM structure of the Saccharomyces cerevisiae small-subunit processome at an overall resolution of 3.8 Å, which provides an essentially complete near-atomic model of this assembly. In this nucleolar superstructure, 51 ribosome-assembly factors and two RNAs encapsulate the 18S rRNA precursor and 15 ribosomal proteins in a state that precedes pre-rRNA cleavage at site A1. Extended flexible proteins are employed to connect distant sites in this particle. Molecular mimicry and steric hindrance, as well as protein- and RNA-mediated RNA remodeling, are used in a concerted fashion to prevent the premature formation of the central pseudoknot and its surrounding elements within the small ribosomal subunit.
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Affiliation(s)
- Jonas Barandun
- Laboratory of Protein and Nucleic Acid Chemistry, The Rockefeller University, New York, New York, USA
| | - Malik Chaker-Margot
- Laboratory of Protein and Nucleic Acid Chemistry, The Rockefeller University, New York, New York, USA
- Tri-Institutional Training Program in Chemical Biology, The Rockefeller University, New York, New York, USA
| | - Mirjam Hunziker
- Laboratory of Protein and Nucleic Acid Chemistry, The Rockefeller University, New York, New York, USA
| | - Kelly R Molloy
- Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York, USA
| | - Brian T Chait
- Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York, USA
| | - Sebastian Klinge
- Laboratory of Protein and Nucleic Acid Chemistry, The Rockefeller University, New York, New York, USA
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31
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32
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Matelska D, Steczkiewicz K, Ginalski K. Comprehensive classification of the PIN domain-like superfamily. Nucleic Acids Res 2017; 45:6995-7020. [PMID: 28575517 PMCID: PMC5499597 DOI: 10.1093/nar/gkx494] [Citation(s) in RCA: 60] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2017] [Accepted: 05/24/2017] [Indexed: 12/21/2022] Open
Abstract
PIN-like domains constitute a widespread superfamily of nucleases, diverse in terms of the reaction mechanism, substrate specificity, biological function and taxonomic distribution. Proteins with PIN-like domains are involved in central cellular processes, such as DNA replication and repair, mRNA degradation, transcription regulation and ncRNA maturation. In this work, we identify and classify the most complete set of PIN-like domains to provide the first comprehensive analysis of sequence–structure–function relationships within the whole PIN domain-like superfamily. Transitive sequence searches using highly sensitive methods for remote homology detection led to the identification of several new families, including representatives of Pfam (DUF1308, DUF4935) and CDD (COG2454), and 23 other families not classified in the public domain databases. Further sequence clustering revealed relationships between individual sequence clusters and showed heterogeneity within some families, suggesting a possible functional divergence. With five structural groups, 70 defined clusters, over 100,000 proteins, and broad biological functions, the PIN domain-like superfamily constitutes one of the largest and most diverse nuclease superfamilies. Detailed analyses of sequences and structures, domain architectures, and genomic contexts allowed us to predict biological function of several new families, including new toxin-antitoxin components, proteins involved in tRNA/rRNA maturation and transcription/translation regulation.
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Affiliation(s)
- Dorota Matelska
- University of Warsaw, CeNT, Laboratory of Bioinformatics and Systems Biology, Zwirki i Wigury 93, 02-089 Warsaw, Poland
| | - Kamil Steczkiewicz
- University of Warsaw, CeNT, Laboratory of Bioinformatics and Systems Biology, Zwirki i Wigury 93, 02-089 Warsaw, Poland
| | - Krzysztof Ginalski
- University of Warsaw, CeNT, Laboratory of Bioinformatics and Systems Biology, Zwirki i Wigury 93, 02-089 Warsaw, Poland
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33
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Tomecki R, Sikorski PJ, Zakrzewska-Placzek M. Comparison of preribosomal RNA processing pathways in yeast, plant and human cells - focus on coordinated action of endo- and exoribonucleases. FEBS Lett 2017; 591:1801-1850. [PMID: 28524231 DOI: 10.1002/1873-3468.12682] [Citation(s) in RCA: 79] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2017] [Revised: 05/14/2017] [Accepted: 05/15/2017] [Indexed: 12/17/2022]
Abstract
Proper regulation of ribosome biosynthesis is mandatory for cellular adaptation, growth and proliferation. Ribosome biogenesis is the most energetically demanding cellular process, which requires tight control. Abnormalities in ribosome production have severe consequences, including developmental defects in plants and genetic diseases (ribosomopathies) in humans. One of the processes occurring during eukaryotic ribosome biogenesis is processing of the ribosomal RNA precursor molecule (pre-rRNA), synthesized by RNA polymerase I, into mature rRNAs. It must not only be accurate but must also be precisely coordinated with other phenomena leading to the synthesis of functional ribosomes: RNA modification, RNA folding, assembly with ribosomal proteins and nucleocytoplasmic RNP export. A multitude of ribosome biogenesis factors ensure that these events take place in a correct temporal order. Among them are endo- and exoribonucleases involved in pre-rRNA processing. Here, we thoroughly present a wide spectrum of ribonucleases participating in rRNA maturation, focusing on their biochemical properties, regulatory mechanisms and substrate specificity. We also discuss cooperation between various ribonucleolytic activities in particular stages of pre-rRNA processing, delineating major similarities and differences between three representative groups of eukaryotes: yeast, plants and humans.
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Affiliation(s)
- Rafal Tomecki
- Laboratory of RNA Biology and Functional Genomics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.,Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Poland
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34
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Senissar M, Manav MC, Brodersen DE. Structural conservation of the PIN domain active site across all domains of life. Protein Sci 2017; 26:1474-1492. [PMID: 28508407 DOI: 10.1002/pro.3193] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2017] [Revised: 05/08/2017] [Accepted: 05/08/2017] [Indexed: 01/26/2023]
Abstract
The PIN (PilT N-terminus) domain is a compact RNA-binding protein domain present in all domains of life. This 120-residue domain consists of a central and parallel β sheet surrounded by α helices, which together organize 4-5 acidic residues in an active site that binds one or more divalent metal ions and in many cases has endoribonuclease activity. In bacteria and archaea, the PIN domain is primarily associated with toxin-antitoxin loci, consisting of a toxin (the PIN domain nuclease) and an antitoxin that inhibits the function of the toxin under normal growth conditions. During nutritional or antibiotic stress, the antitoxin is proteolytically degraded causing activation of the PIN domain toxin leading to a dramatic reprogramming of cellular metabolism to cope with the new situation. In eukaryotes, PIN domains are commonly found as parts of larger proteins and are involved in a range of processes involving RNA cleavage, including ribosomal RNA biogenesis and nonsense-mediated mRNA decay. In this review, we provide a comprehensive overview of the structural characteristics of the PIN domain and compare PIN domains from all domains of life in terms of structure, active site architecture, and activity.
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Affiliation(s)
- M Senissar
- Centre for Bacterial Stress Response and Persistence, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10c, Aarhus, 8000, Denmark
| | - M C Manav
- Centre for Bacterial Stress Response and Persistence, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10c, Aarhus, 8000, Denmark
| | - D E Brodersen
- Centre for Bacterial Stress Response and Persistence, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10c, Aarhus, 8000, Denmark
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35
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Zhai B, DuPrez K, Doukov TI, Li H, Huang M, Shang G, Ni J, Gu L, Shen Y, Fan L. Structure and Function of a Novel ATPase that Interacts with Holliday Junction Resolvase Hjc and Promotes Branch Migration. J Mol Biol 2017; 429:1009-1029. [PMID: 28238763 PMCID: PMC5565510 DOI: 10.1016/j.jmb.2017.02.016] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2016] [Revised: 01/25/2017] [Accepted: 02/19/2017] [Indexed: 11/15/2022]
Abstract
Holliday junction (HJ) is a hallmark intermediate in DNA recombination and must be processed by dissolution (for double HJ) or resolution to ensure genome stability. Although HJ resolvases have been identified in all domains of life, there is a long-standing effort to search in prokaryotes and eukarya for proteins promoting HJ migration. Here, we report the structural and functional characterization of a novel ATPase, Sulfolobus islandicusPilT N-terminal-domain-containing ATPase (SisPINA), encoded by the gene adjacent to the resolvase Hjc coding gene. PINA is conserved in archaea and vital for S. islandicus viability. Purified SisPINA forms hexameric rings in the crystalline state and in solution, similar to the HJ migration helicase RuvB in Gram-negative bacteria. Structural analysis suggests that ATP binding and hydrolysis cause conformational changes in SisPINA to drive branch migration. Further studies reveal that SisPINA interacts with SisHjc and coordinates HJ migration and cleavage.
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Affiliation(s)
- Binyuan Zhai
- State Key Laboratory of Microbial Technology, Shandong University, 27 Shanda Nan Road, Jinan 250100, PR China
| | - Kevin DuPrez
- Department of Biochemistry, University of California, Riverside, CA 92521, USA
| | - Tzanko I Doukov
- Macromolecular Crystallography Group, Stanford Synchrotron Radiation Light Source, SLAC National Accelerator Laboratory, Stanford University, Stanford, CA 94309, USA
| | - Huan Li
- State Key Laboratory of Microbial Technology, Shandong University, 27 Shanda Nan Road, Jinan 250100, PR China
| | - Mengting Huang
- State Key Laboratory of Microbial Technology, Shandong University, 27 Shanda Nan Road, Jinan 250100, PR China
| | - Guijun Shang
- State Key Laboratory of Microbial Technology, Shandong University, 27 Shanda Nan Road, Jinan 250100, PR China
| | - Jinfeng Ni
- State Key Laboratory of Microbial Technology, Shandong University, 27 Shanda Nan Road, Jinan 250100, PR China
| | - Lichuan Gu
- State Key Laboratory of Microbial Technology, Shandong University, 27 Shanda Nan Road, Jinan 250100, PR China
| | - Yulong Shen
- State Key Laboratory of Microbial Technology, Shandong University, 27 Shanda Nan Road, Jinan 250100, PR China.
| | - Li Fan
- Department of Biochemistry, University of California, Riverside, CA 92521, USA.
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36
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Kala S, Mehta V, Yip CW, Moshiri H, Najafabadi HS, Ma R, Nikpour N, Zimmer SL, Salavati R. The interaction of a Trypanosoma brucei KH-domain protein with a ribonuclease is implicated in ribosome processing. Mol Biochem Parasitol 2016; 211:94-103. [PMID: 27965085 DOI: 10.1016/j.molbiopara.2016.12.003] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2016] [Revised: 12/08/2016] [Accepted: 12/09/2016] [Indexed: 12/24/2022]
Abstract
Ribosomal RNA maturation is best understood in yeast. While substantial efforts have been made to explore parts of these essential pathways in animals, the similarities and uniquenesses of rRNA maturation factors in non-Opisthokonts remain largely unexplored. Eukaryotic ribosome synthesis requires the coordinated activities of hundreds of Assembly Factors (AFs) that transiently associate with pre-ribosomes, many of which are essential. Pno1 and Nob1 are two of six AFs that are required for the cytoplasmic maturation of the 20S pre-rRNA to 18S rRNA in yeast where it has been almost exclusively analyzed. Specifically, Nob1 ribonucleolytic activity generates the mature 3'-end of 18S rRNA. We identified putative Pno1 and Nob1 homologues in the protist Trypanosoma brucei, named TbPNO1 and TbNOB1, and set out to explore their rRNA maturation role further as they are both essential for normal growth. TbPNO1 is a nuclear protein with limited cytosolic localization relative to its yeast homologue. Like in yeast, it interacts directly with TbNOB1, with indications of associations with a larger AF-containing complex. Interestingly, in the absence of TbPNO1, TbNOB1 exhibits non-specific degradation activity on RNA substrates, and its cleavage activity becomes specific only in the presence of TbPNO1, suggesting that TbPNO1-TbNOB1 interaction is essential for regulation and site-specificity of TbNOB1 activity. These results highlight a conserved role of the TbPNO1-TbNOB1 complex in 18S rRNA maturation across eukaryotes; yet reveal a novel role of their interaction in regulation of TbNOB1 enzymatic activity.
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Affiliation(s)
- Smriti Kala
- Institute of Parasitology, McGill University, Quebec, H9X3V9, Canada
| | - Vaibhav Mehta
- Institute of Parasitology, McGill University, Quebec, H9X3V9, Canada; Department of Biochemistry, McGill University, McIntyre Medical Building, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada
| | - Chun Wai Yip
- Institute of Parasitology, McGill University, Quebec, H9X3V9, Canada
| | - Houtan Moshiri
- Institute of Parasitology, McGill University, Quebec, H9X3V9, Canada; Department of Biochemistry, McGill University, McIntyre Medical Building, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada
| | | | - Ruoyu Ma
- Institute of Parasitology, McGill University, Quebec, H9X3V9, Canada
| | - Najmeh Nikpour
- Institute of Parasitology, McGill University, Quebec, H9X3V9, Canada
| | - Sara L Zimmer
- Department of Biomedical Sciences, University of Minnesota Medical School, Duluth, MN, 55812, USA
| | - Reza Salavati
- Institute of Parasitology, McGill University, Quebec, H9X3V9, Canada; Department of Biochemistry, McGill University, McIntyre Medical Building, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada.
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37
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Peña C, Schütz S, Fischer U, Chang Y, Panse VG. Prefabrication of a ribosomal protein subcomplex essential for eukaryotic ribosome formation. eLife 2016; 5. [PMID: 27929371 PMCID: PMC5148605 DOI: 10.7554/elife.21755] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2016] [Accepted: 11/29/2016] [Indexed: 01/21/2023] Open
Abstract
Spatial clustering of ribosomal proteins (r-proteins) through tertiary interactions is a striking structural feature of the eukaryotic ribosome. However, the functional importance of these intricate inter-connections, and how they are established is currently unclear. Here, we reveal that a conserved ATPase, Fap7, organizes interactions between neighboring r-proteins uS11 and eS26 prior to their delivery to the earliest ribosome precursor, the 90S. In vitro, uS11 only when bound to Fap7 becomes competent to recruit eS26 through tertiary contacts found between these r-proteins on the mature ribosome. Subsequently, Fap7 ATPase activity unloads the uS11:eS26 subcomplex onto its rRNA binding site, and therefore ensures stoichiometric integration of these r-proteins into the 90S. Fap7-depletion in vivo renders uS11 susceptible to proteolysis, and precludes eS26 incorporation into the 90S. Thus, prefabrication of a native-like r-protein subcomplex drives efficient and accurate construction of the eukaryotic ribosome. DOI:http://dx.doi.org/10.7554/eLife.21755.001
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Affiliation(s)
- Cohue Peña
- Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.,Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland
| | - Sabina Schütz
- Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland
| | - Ute Fischer
- Institute of Biochemistry, ETH Zurich, Zurich, Switzerland
| | - Yiming Chang
- Institute of Biochemistry, ETH Zurich, Zurich, Switzerland
| | - Vikram G Panse
- Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland
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38
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Larburu N, Montellese C, O'Donohue MF, Kutay U, Gleizes PE, Plisson-Chastang C. Structure of a human pre-40S particle points to a role for RACK1 in the final steps of 18S rRNA processing. Nucleic Acids Res 2016; 44:8465-78. [PMID: 27530427 PMCID: PMC5041492 DOI: 10.1093/nar/gkw714] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2016] [Revised: 07/28/2016] [Accepted: 08/06/2016] [Indexed: 01/24/2023] Open
Abstract
Synthesis of ribosomal subunits in eukaryotes is a complex and tightly regulated process that has been mostly characterized in yeast. The discovery of a growing number of diseases linked to defects in ribosome biogenesis calls for a deeper understanding of these mechanisms and of the specificities of human ribosome maturation. We present the 19 Å resolution cryo-EM reconstruction of a cytoplasmic precursor to the human small ribosomal subunit, purified by using the tagged ribosome biogenesis factor LTV1 as bait. Compared to yeast pre-40S particles, this first three-dimensional structure of a human 40S subunit precursor shows noticeable differences with respect to the position of ribosome biogenesis factors and uncovers the early deposition of the ribosomal protein RACK1 during subunit maturation. Consistently, RACK1 is required for efficient processing of the 18S rRNA 3'-end, which might be related to its role in translation initiation. This first structural analysis of a human pre-ribosomal particle sets the grounds for high-resolution studies of conformational transitions accompanying ribosomal subunit maturation.
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MESH Headings
- Conserved Sequence
- Cryoelectron Microscopy
- Cytoplasm/metabolism
- GTP-Binding Proteins/metabolism
- HEK293 Cells
- HeLa Cells
- Humans
- Models, Molecular
- Neoplasm Proteins/metabolism
- Organelle Biogenesis
- Protein Binding
- RNA Processing, Post-Transcriptional/genetics
- RNA, Ribosomal, 18S/genetics
- Receptors for Activated C Kinase
- Receptors, Cell Surface/metabolism
- Ribosomal Proteins/metabolism
- Ribosome Subunits, Small, Eukaryotic/chemistry
- Ribosome Subunits, Small, Eukaryotic/metabolism
- Ribosome Subunits, Small, Eukaryotic/ultrastructure
- Saccharomyces cerevisiae/metabolism
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Affiliation(s)
- Natacha Larburu
- Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, France
| | | | - Marie-Françoise O'Donohue
- Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, France
| | - Ulrike Kutay
- Institut für Biochemie, ETH Zürich, CH-8093 Zurich, Switzerland
| | - Pierre-Emmanuel Gleizes
- Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, France
| | - Célia Plisson-Chastang
- Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, France
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39
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Winther K, Tree JJ, Tollervey D, Gerdes K. VapCs of Mycobacterium tuberculosis cleave RNAs essential for translation. Nucleic Acids Res 2016; 44:9860-9871. [PMID: 27599842 PMCID: PMC5175351 DOI: 10.1093/nar/gkw781] [Citation(s) in RCA: 100] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2016] [Revised: 08/21/2016] [Accepted: 08/25/2016] [Indexed: 01/16/2023] Open
Abstract
The major human pathogen Mycobacterium tuberculosis can survive in the host organism for decades without causing symptoms. A large cohort of Toxin–Antitoxin (TA) modules contribute to this persistence. Of these, 48 TA modules belong to the vapBC (virulence associated protein) gene family. VapC toxins are PIN domain endonucleases that, in enterobacteria, inhibit translation by site-specific cleavage of initiator tRNA. In contrast, VapC20 of M. tuberculosis inhibits translation by site-specific cleavage of the universally conserved Sarcin-Ricin loop (SRL) in 23S rRNA. Here we identify the cellular targets of 12 VapCs from M. tuberculosis by applying UV-crosslinking and deep sequencing. Remarkably, these VapCs are all endoribonucleases that cleave RNAs essential for decoding at the ribosomal A-site. Eleven VapCs cleave specific tRNAs while one exhibits SRL cleavage activity. These findings suggest that multiple vapBC modules contribute to the survival of M. tuberculosis in its human host by reducing the level of translation.
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Affiliation(s)
- Kristoffer Winther
- Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark .,Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Richardson Road, NE2 4AX, Newcastle upon Tyne, UK
| | - Jai J Tree
- School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2033, Australia
| | - David Tollervey
- Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, King's Buildings, Edinburgh EH9 3BF, UK
| | - Kenn Gerdes
- Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark .,Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Richardson Road, NE2 4AX, Newcastle upon Tyne, UK
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40
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Bai D, Zhang J, Li T, Hang R, Liu Y, Tian Y, Huang D, Qu L, Cao X, Ji J, Zheng X. The ATPase hCINAP regulates 18S rRNA processing and is essential for embryogenesis and tumour growth. Nat Commun 2016; 7:12310. [PMID: 27477389 PMCID: PMC4974663 DOI: 10.1038/ncomms12310] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2016] [Accepted: 06/20/2016] [Indexed: 12/29/2022] Open
Abstract
Dysfunctions in ribosome biogenesis cause developmental defects and increased cancer susceptibility; however, the connection between ribosome assembly and tumorigenesis remains unestablished. Here we show that hCINAP (also named AK6) is required for human 18S rRNA processing and 40S subunit assembly. Homozygous CINAP−/− mice show embryonic lethality. The heterozygotes are viable and show defects in 18S rRNA processing, whereas no delayed cell growth is observed. However, during rapid growth, CINAP haploinsufficiency impairs protein synthesis. Consistently, hCINAP depletion in fast-growing cancer cells inhibits ribosome assembly and abolishes tumorigenesis. These data demonstrate that hCINAP reduction is a specific rate-limiting controller during rapid growth. Notably, hCINAP is highly expressed in cancers and correlated with a worse prognosis. Genome-wide polysome profiling shows that hCINAP selectively modulates cancer-associated translatome to promote malignancy. Our results connect the role of hCINAP in ribosome assembly with tumorigenesis. Modulation of hCINAP expression may be a promising target for cancer therapy. Perturbations in ribosome biogenesis affect development and increase cancer susceptibility. Here, the authors show that hCINAP is required for 18S rRNA processing, is highly expressed in cancers, and promotes cancer cell growth by upregulating the translation of cancer-associated genes.
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Affiliation(s)
- Dongmei Bai
- State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China.,Department of Biochemistry and Molecular Biology, School of Life Sciences, Peking University, Yiheyuan Road No. 5, Beijing 100871, China
| | - Jinfang Zhang
- State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China.,Department of Biochemistry and Molecular Biology, School of Life Sciences, Peking University, Yiheyuan Road No. 5, Beijing 100871, China
| | - Tingting Li
- State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China.,Department of Biochemistry and Molecular Biology, School of Life Sciences, Peking University, Yiheyuan Road No. 5, Beijing 100871, China
| | - Runlai Hang
- State key Laboratory of Plant Genetics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.,College of Life Sciences, University of the Chinese Academy of Sciences, Beijing 100039, China
| | - Yong Liu
- State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China.,Department of Biochemistry and Molecular Biology, School of Life Sciences, Peking University, Yiheyuan Road No. 5, Beijing 100871, China
| | - Yonglu Tian
- Department of Biochemistry and Molecular Biology, School of Life Sciences, Peking University, Yiheyuan Road No. 5, Beijing 100871, China
| | - Dadu Huang
- Department of Biochemistry and Molecular Biology, School of Life Sciences, Peking University, Yiheyuan Road No. 5, Beijing 100871, China
| | - Linglong Qu
- State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China.,Department of Biochemistry and Molecular Biology, School of Life Sciences, Peking University, Yiheyuan Road No. 5, Beijing 100871, China
| | - Xiaofeng Cao
- State key Laboratory of Plant Genetics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.,College of Life Sciences, University of the Chinese Academy of Sciences, Beijing 100039, China
| | - Jiafu Ji
- Key Laboratory of Carcinogenesis and Translational Research, Department of Gastrointestinal Surgery, Peking University Caner Hospital and Institute, Beijing 100142, China
| | - Xiaofeng Zheng
- State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China.,Department of Biochemistry and Molecular Biology, School of Life Sciences, Peking University, Yiheyuan Road No. 5, Beijing 100871, China
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41
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Tomecki R, Labno A, Drazkowska K, Cysewski D, Dziembowski A. hUTP24 is essential for processing of the human rRNA precursor at site A1, but not at site A0. RNA Biol 2016; 12:1010-29. [PMID: 26237581 DOI: 10.1080/15476286.2015.1073437] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Production of ribosomes relies on more than 200 accessory factors to ensure the proper sequence of steps and faultless assembly of ribonucleoprotein machinery. Among trans-acting factors are numerous enzymes, including ribonucleases responsible for processing the large rRNA precursor synthesized by RNA polymerase I that encompasses sequences corresponding to mature 18S, 5.8S, and 25/28S rRNA. In humans, the identity of most enzymes responsible for individual processing steps, including endoribonucleases that cleave pre-rRNA at specific sites within regions flanking and separating mature rRNA, remains largely unknown. Here, we investigated the role of hUTP24 in rRNA maturation in human cells. hUTP24 is a human homolog of the Saccharomyces cerevisiae putative PIN domain-containing endoribonuclease Utp24 (yUtp24), which was suggested to participate in the U3 snoRNA-dependent processing of yeast pre-rRNA at sites A0, A1, and A2. We demonstrate that hUTP24 interacts to some extent with proteins homologous to the components of the yeast small subunit (SSU) processome. Moreover, mutation in the putative catalytic site of hUTP24 results in slowed growth of cells and reduced metabolic activity. These effects are associated with a defect in biogenesis of the 40S ribosomal subunit, which results from decreased amounts of 18S rRNA as a consequence of inaccurate pre-rRNA processing at the 5'-end of the 18S rRNA segment (site A1). Interestingly, and in contrast to yeast, site A0 located upstream of A1 is efficiently processed upon UTP24 dysfunction. Finally, hUTP24 inactivation leads to aberrant processing of 18S rRNA 2 nucleotides downstream of the normal A1 cleavage site.
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Affiliation(s)
- Rafal Tomecki
- a Institute of Biochemistry and Biophysics; Polish Academy of Sciences ; Warsaw , Poland.,b Institute of Genetics and Biotechnology; Faculty of Biology; University of Warsaw ; Warsaw , Poland
| | - Anna Labno
- a Institute of Biochemistry and Biophysics; Polish Academy of Sciences ; Warsaw , Poland.,b Institute of Genetics and Biotechnology; Faculty of Biology; University of Warsaw ; Warsaw , Poland
| | - Karolina Drazkowska
- a Institute of Biochemistry and Biophysics; Polish Academy of Sciences ; Warsaw , Poland.,b Institute of Genetics and Biotechnology; Faculty of Biology; University of Warsaw ; Warsaw , Poland
| | - Dominik Cysewski
- a Institute of Biochemistry and Biophysics; Polish Academy of Sciences ; Warsaw , Poland.,b Institute of Genetics and Biotechnology; Faculty of Biology; University of Warsaw ; Warsaw , Poland
| | - Andrzej Dziembowski
- a Institute of Biochemistry and Biophysics; Polish Academy of Sciences ; Warsaw , Poland.,b Institute of Genetics and Biotechnology; Faculty of Biology; University of Warsaw ; Warsaw , Poland
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42
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Gao X, Wang J, Bai W, Ji W, Wang L. NOB1 silencing inhibits the growth and metastasis of laryngeal cancer cells through the regulation of JNK signaling pathway. Oncol Rep 2016; 35:3313-20. [PMID: 27035645 DOI: 10.3892/or.2016.4707] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2015] [Accepted: 01/05/2016] [Indexed: 11/06/2022] Open
Abstract
Nin one binding protein (NOB1) plays important roles in the synthesis and degradation of proteins, thus having effects on the cellular process. In the present study, the expression level of NOB1 in laryngeal cancer patients was detected by quantitative PCR and western blotting, and the effect of NOB1 on growth and metastasis of laryngeal cancer cells was explored. Silence of NOB1 was found to inhibit the proliferation of laryngeal cancer cells, arrest cell cycle and induce cell apoptosis. NOB1 silence was also found to inhibit the migration and invasion of laryngeal cancer cells and to downregulate the protein levels of matrix metalloproteinases (MMPs)-2 and MMP-9. Further mechanism study revealed that the JNK signaling pathway was involved in the function of NOB1. Our present results suggest that NOB1 plays an oncogenic role in laryngeal cancer cells through the regulation of JNK signaling pathway, and lays a theoretical foundation for further exploration of NOB1.
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Affiliation(s)
- Xin Gao
- Department of Otorhinolaryngology-Head and Neck Surgery, Shengjing Hospital of China Medical University, Heping, Shenyang, Liaoning 110004, P.R. China
| | - Jin Wang
- Department of Otorhinolaryngology-Head and Neck Surgery, Shengjing Hospital of China Medical University, Heping, Shenyang, Liaoning 110004, P.R. China
| | - Weiliang Bai
- Department of Otorhinolaryngology-Head and Neck Surgery, Shengjing Hospital of China Medical University, Heping, Shenyang, Liaoning 110004, P.R. China
| | - Wenyue Ji
- Department of Otorhinolaryngology-Head and Neck Surgery, Shengjing Hospital of China Medical University, Heping, Shenyang, Liaoning 110004, P.R. China
| | - Liping Wang
- Department of Otorhinolaryngology-Head and Neck Surgery, Shengjing Hospital of China Medical University, Heping, Shenyang, Liaoning 110004, P.R. China
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43
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Yin J, Wang J, Jiang Y, Wang L, Wu H, Liu H. Downregulation of NOB1 inhibits proliferation and promotes apoptosis in human oral squamous cell carcinoma. Oncol Rep 2015; 34:3077-87. [PMID: 26370469 DOI: 10.3892/or.2015.4271] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2015] [Accepted: 07/09/2015] [Indexed: 11/06/2022] Open
Abstract
NIN1/RPN12 binding protein 1 homolog (NOB1) facilitates the maturation of the 20S proteasome and is then degraded by 26S proteasome to complete 26S proteasome biogenesis. It also accompanies the pre-40S ribosomes during nuclear export and is cleaved at D-site of 20S pre-rRNA to form mature 18S rRNA in growing cells. NOB1 was reported to be involved in the development of several types of cancer. However, the role of NOB1 in oral squamous cell carcinoma (OSCC) has not been addressed. In the present study, the expression of NOB1 in 50 OSCC patients with different genders, ages, TNM and pathological grades was detected using immunohistochemistry and western blotting. A loss-of‑function study was carried out by the lentivirus‑mediated siRNA knockdown of NOB1 in the CAL27 and TCA-8113 OSCC cell lines. The results showed that, NOB1 expression increased with pathological grades. In the CAL27 and TCA-8113 cell lines, knockdown of NOB1 in OSCC cells decreased cell proliferation, colony formation, increased cell apoptosis and also induced cell cycle arrest in the S phase. The results suggested that NOB1 is important in OSCC development and serves as a candidate indicator of aggressiveness and a therapeutic target of OSCC.
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Affiliation(s)
- Jirong Yin
- Dental Institute, Chinese PLA General Hospital, Beijing 100853, P.R. China
| | - Juncheng Wang
- Dental Institute, Chinese PLA General Hospital, Beijing 100853, P.R. China
| | - Yi Jiang
- Dental Institute, Chinese PLA General Hospital, Beijing 100853, P.R. China
| | - Lili Wang
- Dental Institute, Chinese PLA General Hospital, Beijing 100853, P.R. China
| | - Hao Wu
- Dental Institute, Chinese PLA General Hospital, Beijing 100853, P.R. China
| | - Hongchen Liu
- Dental Institute, Chinese PLA General Hospital, Beijing 100853, P.R. China
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44
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HUANG WEIYI, ZHONG WEIQING, XU JUN, SU BENHUA, HUANG GUANGHUI, DU JIAJUN, LIU QI. Lentivirus-mediated gene silencing of NOB1 suppresses non-small cell lung cancer cell proliferation. Oncol Rep 2015; 34:1510-6. [DOI: 10.3892/or.2015.4132] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2015] [Accepted: 06/28/2015] [Indexed: 11/05/2022] Open
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45
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Structure-function analysis of VapB4 antitoxin identifies critical features of a minimal VapC4 toxin-binding module. J Bacteriol 2015; 197:1197-207. [PMID: 25622615 DOI: 10.1128/jb.02508-14] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
UNLABELLED Bacterial toxin-antitoxin systems play a critical role in the regulation of gene expression, leading to developmental changes, reversible dormancy, and cell death. Type II toxin-antitoxin pairs, composed of protein toxins and antitoxins, exist in nearly all bacteria and are classified into six groups on the basis of the structure of the toxins. The VapBC group comprises the most common type II system and, like other toxin-antitoxin systems, functions to elicit dormancy by inhibiting protein synthesis. Activation of toxin function requires protease degradation of the VapB antitoxin, which frees the VapC toxin from the VapBC complex, allowing it to hydrolyze the RNAs required for translation. Generally, type II antitoxins bind with high specificity to their cognate toxins via a toxin-binding domain and endow the complex with DNA-binding specificity via a DNA-binding domain. Despite the ubiquity of VapBC systems and their critical role in the regulation of gene expression, few functional studies have addressed the details of VapB-VapC interactions. Here we report on the results of experiments designed to identify molecular determinants of the specificity of the Mycobacterium tuberculosis VapB4 antitoxin for its cognate VapC4 toxin. The results identify the minimal domain of VapB4 required for this interaction as well as the amino acid side chains required for binding to VapC4. These findings have important implications for the evolution of VapBC toxin-antitoxin systems and their potential as targets of small-molecule protein-protein interaction inhibitors. IMPORTANCE VapBC toxin-antitoxin pairs are the most widespread type II toxin-antitoxin systems in bacteria, where they are thought to play key roles in stress-induced dormancy and the formation of persisters. The VapB antitoxins are critical to these processes because they inhibit the activity of the toxins and provide the DNA-binding specificity that controls the synthesis of both proteins. Despite the importance of VapB antitoxins and the existence of several VapBC crystal structures, little is known about their functional features in vivo. Here we report the findings of the first comprehensive structure-function analysis of a VapB toxin. The results identify the minimal toxin-binding domain, its modular antitoxin function, and the specific amino acid side chains required for its activity.
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46
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He XW, Feng T, Yin QL, Jian YW, Liu T. NOB1 is essential for the survival of RKO colorectal cancer cells. World J Gastroenterol 2015; 21:868-877. [PMID: 25624720 PMCID: PMC4299339 DOI: 10.3748/wjg.v21.i3.868] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/01/2014] [Revised: 06/13/2014] [Accepted: 07/22/2014] [Indexed: 02/06/2023] Open
Abstract
AIM: To determine the role of NOB1, a regulator of cell survival in yeast, in human colorectal cancer cells.
METHODS: Lentivirus-mediated small interfering RNA (siRNA) was used to inhibit NOB1 expression in RKO human colorectal cancer cells in vitro and in vivo in a mouse xenograft model. The in vitro and in vivo knockdown efficacy was determined using both Western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR was also used to analyze the downstream signals following NOB1 knockdown. Cell growth and colony formation assays were used to determine the effect of NOB1 inhibition on RKO proliferation and their ability to form colonies. Endonuclease activity, as evaluated by terminal deoxytransferase-mediated dUTP nick end labeling (TUNEL), and annexin V staining were used to determine the presence of apoptotic cell death prior to and following NOB1 inhibition. Cell cycle analysis was used to determine the effect of NOB1 inhibition on RKO cell cycle. A cDNA microarray was used to determine global differential gene expression following NOB1 knockdown.
RESULTS: Virus-mediated siRNA inhibition of NOB1 resulted in (1) the down-regulation of NOB1 expression in RKO cells for both the mRNA and protein; (2) inhibition of NOB1 expression both in vitro and in vivo experimental systems; (3) cell growth inhibition via significant induction of cell apoptosis, without alteration of the cell cycle distribution; and (4) a significant decrease in the average weight and volume of xenograft tumors in the NOB1-siRNA group compared to the control scr-siRNA group (P = 0.001, P < 0.05). Significantly more apoptosis was detected within tumors in the NOB1-siRNA group than in the control group. Microarray analysis detected 2336 genes potentially regulated by NOB1. Most of these genes are associated with the WNT, cell proliferation, apoptosis, fibroblast growth factor, and angiogenesis signaling pathways, of which BAX and WNT were validated by qRT-PCR. Among them, 1451 probes, representing 963 unique genes, were upregulated; however, 2308 probes, representing 1373 unique genes, were downregulated.
CONCLUSION: NOB1 gene silencing by lentivirus-mediated RNA interference can inhibit tumor growth by inducing apoptosis of cancerous human colorectal cells.
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47
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Burroughs AM, Aravind L. Analysis of two domains with novel RNA-processing activities throws light on the complex evolution of ribosomal RNA biogenesis. Front Genet 2014; 5:424. [PMID: 25566315 PMCID: PMC4275035 DOI: 10.3389/fgene.2014.00424] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2014] [Accepted: 11/19/2014] [Indexed: 11/13/2022] Open
Abstract
Ribosomal biogenesis has been extensively investigated, especially to identify the elusive nucleases and cofactors involved in the complex rRNA processing events in eukaryotes. Large-scale screens in yeast identified two biochemically uncharacterized proteins, TSR3 and TSR4, as being key players required for rRNA maturation. Using multiple computational approaches we identify the conserved domains comprising these proteins and establish sequence and structural features providing novel insights regarding their roles. TSR3 is unified with the DTW domain into a novel superfamily of predicted enzymatic domains, with the balance of the available evidence pointing toward an RNase role with the archaeo-eukaryotic TSR3 proteins processing rRNA and the bacterial versions potentially processing tRNA. TSR4, its other eukaryotic homologs PDCD2/rp-8, PDCD2L, Zfrp8, and trus, the predominantly bacterial DUF1963 proteins, and other uncharacterized proteins are unified into a new domain superfamily, which arose from an ancient duplication event of a strand-swapped, dimer-forming all-beta unit. We identify conserved features mediating protein-protein interactions (PPIs) and propose a potential chaperone-like function. While contextual evidence supports a conserved role in ribosome biogenesis for the eukaryotic TSR4-related proteins, there is no evidence for such a role for the bacterial versions. Whereas TSR3-related proteins can be traced to the last universal common ancestor (LUCA) with a well-supported archaeo-eukaryotic branch, TSR4-related proteins of eukaryotes are derived from within the bacterial radiation of this superfamily, with archaea entirely lacking them. This provides evidence for “systems admixture,” which followed the early endosymbiotic event, playing a key role in the emergence of the uniquely eukaryotic ribosome biogenesis process.
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Affiliation(s)
- A Maxwell Burroughs
- National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health Bethesda, MD, USA
| | - L Aravind
- National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health Bethesda, MD, USA
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48
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Hamilton B, Manzella A, Schmidt K, DiMarco V, Butler JS. Analysis of non-typeable Haemophilous influenzae VapC1 mutations reveals structural features required for toxicity and flexibility in the active site. PLoS One 2014; 9:e112921. [PMID: 25391136 PMCID: PMC4229260 DOI: 10.1371/journal.pone.0112921] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2014] [Accepted: 10/21/2014] [Indexed: 02/06/2023] Open
Abstract
Bacteria have evolved mechanisms that allow them to survive in the face of a variety of stresses including nutrient deprivation, antibiotic challenge and engulfment by predator cells. A switch to dormancy represents one strategy that reduces energy utilization and can render cells resistant to compounds that kill growing bacteria. These persister cells pose a problem during treatment of infections with antibiotics, and dormancy mechanisms may contribute to latent infections. Many bacteria encode toxin-antitoxin (TA) gene pairs that play an important role in dormancy and the formation of persisters. VapBC gene pairs comprise the largest of the Type II TA systems in bacteria and they produce a VapC ribonuclease toxin whose activity is inhibited by the VapB antitoxin. Despite the importance of VapBC TA pairs in dormancy and persister formation, little information exists on the structural features of VapC proteins required for their toxic function in vivo. Studies reported here identified 17 single mutations that disrupt the function of VapC1 from non-typeable H. influenzae in vivo. 3-D modeling suggests that side chains affected by many of these mutations sit near the active site of the toxin protein. Phylogenetic comparisons and secondary mutagenesis indicate that VapC1 toxicity requires an alternative active site motif found in many proteobacteria. Expression of the antitoxin VapB1 counteracts the activity of VapC1 mutants partially defective for toxicity, indicating that the antitoxin binds these mutant proteins in vivo. These findings identify critical chemical features required for the biological function of VapC toxins and PIN-domain proteins.
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Affiliation(s)
- Brooke Hamilton
- Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, United States of America
| | - Alexander Manzella
- Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, United States of America
| | - Karyn Schmidt
- Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York, United States of America
| | - Victoria DiMarco
- Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, United States of America
| | - J. Scott Butler
- Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, United States of America
- Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York, United States of America
- Center for RNA Biology, University of Rochester Medical Center, Rochester, New York, United States of America
- * E-mail:
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49
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Turowski TW, Lebaron S, Zhang E, Peil L, Dudnakova T, Petfalski E, Granneman S, Rappsilber J, Tollervey D. Rio1 mediates ATP-dependent final maturation of 40S ribosomal subunits. Nucleic Acids Res 2014; 42:12189-99. [PMID: 25294836 PMCID: PMC4231747 DOI: 10.1093/nar/gku878] [Citation(s) in RCA: 69] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
During the last step in 40S ribosome subunit biogenesis, the PIN-domain endonuclease Nob1 cleaves the 20S pre-rRNA at site D, to form the mature 18S rRNAs. Here we report that cleavage occurs in particles that have largely been stripped of previously characterized pre-40S components, but retain the endonuclease Nob1, its binding partner Pno1 (Dim2) and the atypical ATPase Rio1. Within the Rio1-associated pre-40S particles, in vitro pre-rRNA cleavage was strongly stimulated by ATP and required nucleotide binding by Rio1. In vivo binding sites for Rio1, Pno1 and Nob1 were mapped by UV cross-linking in actively growing cells. Nob1 and Pno1 bind overlapping regions within the internal transcribed spacer 1, and both bind directly over cleavage site D. Binding sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2. Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assemble into these complexes, whereas Pno1 appeared to be depleted relative to Nob1. We speculate that Rio1-mediated dissociation of Pno1 from cleavage site D is the trigger for final 18S rRNA maturation.
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Affiliation(s)
- Tomasz W Turowski
- Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK
| | - Simon Lebaron
- Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK
| | - Elodie Zhang
- Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK
| | - Lauri Peil
- Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK
| | - Tatiana Dudnakova
- Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK
| | - Elisabeth Petfalski
- Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK
| | - Sander Granneman
- Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK
| | - Juri Rappsilber
- Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK
| | - David Tollervey
- Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK
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Meng W, Wang PS, Liu J, Xue S, Wang GM, Meng XY, Chen G. Adenovirus-mediated siRNA targeting NOB1 inhibits tumor growth and enhances radiosensitivity of human papillary thyroid carcinoma in vitro and in vivo. Oncol Rep 2014; 32:2411-20. [PMID: 25231838 DOI: 10.3892/or.2014.3483] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2014] [Accepted: 08/12/2014] [Indexed: 11/05/2022] Open
Abstract
NIN1/RPN12 binding protein 1 homolog (NOB1), a ribosome assembly factor, plays critical roles in tumor progression and development. Previously, we reported that overexpression of NOB1 is correlated with the prognosis of patients with papillary thyroid carcinoma (PTC). Little is known, however, concerning its role in PTC. The aims of the present study were to investigate the association of NOB1 expression with tumor growth and radiosensitivity of human PTC. A recombinant adenovirus expression vector carrying NOB1 was constructed and then infected into the human PTC cell line TPC-1. Cell proliferation, cell cycle distribution, apoptosis, migration and invasion in vitro and tumor growth in vivo were determined after downregulation of NOB1 by RNAi. Additionally, the in vitro and in vivo radiosensitivity of PTC cells was determined by clonogenic cell survival assay and a mouse xenograft model, respectively. The results showed that downregulation of NOB1 expression using RNAi in TPC-1 cells significantly inhibited cell proliferation, migration and invasion and induced cell apoptosis in vitro, and suppressed tumor growth in vivo, as well as enhanced the in vitro and in vivo radiosensitivity of PTC cells. Moreover, our results also showed that downregulation of NOB1 was able to significantly activate constitutive phosphorylation of p38 MAPK, which might contribute to the inhibition of PTC cell growth. These findings suggest that NOB1 may be a potential therapeutic target for the treatment of PTC.
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Affiliation(s)
- Wei Meng
- Department of Thyroid Surgery, The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
| | - Pei-Song Wang
- Department of Thyroid Surgery, The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
| | - Jia Liu
- Department of Thyroid Surgery, The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
| | - Shuai Xue
- Department of Thyroid Surgery, The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
| | - Gui-Min Wang
- Department of Thyroid Surgery, The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
| | - Xian-Ying Meng
- Department of Thyroid Surgery, The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
| | - Guang Chen
- Department of Thyroid Surgery, The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
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