|
1
|
Miura F, Shibata Y, Miura M, Inatomi K, Suzuki Y, Ito T. Identification of an enzyme with strong single-stranded DNA ligation activity and its application for sequencing. Nucleic Acids Res 2025; 53:gkaf054. [PMID: 39883008 PMCID: PMC11780859 DOI: 10.1093/nar/gkaf054] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2024] [Revised: 01/14/2025] [Accepted: 01/21/2025] [Indexed: 01/31/2025] Open
Abstract
An enzyme with strong single-stranded DNA (ssDNA) ligation activity would be advantageous for many molecular biology applications. However, currently available enzymes exhibit only limited activity. Here, we identified an enzyme with strong ssDNA ligation activity upon searching the databases for proteins homologous to TS2126 RNA ligase, the known enzyme with the highest yet limited ssDNA ligation activity. A ligase from Thermus phage phiLo (GenBank: AYJ73970) or Tph ssDNA ligase (SDL) depicted higher ssDNA ligation activity than TS2126 RNA ligase. This study suggests that SDL could be useful for various applications including sequencing for library preparation.
Collapse
Affiliation(s)
- Fumihito Miura
- Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Maidashi 3-1-1, Higashi-Ku, Fukuoka 812-8582, Japan
- Life Science Data Research Center, Graduate School of Frontier Sciences, the University of Tokyo, Kashiwanoha 5-1-5, Kashiwa, Chiba277-8561, Japan
| | - Yukiko Shibata
- Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Maidashi 3-1-1, Higashi-Ku, Fukuoka 812-8582, Japan
| | - Miki Miura
- Life Science Data Research Center, Graduate School of Frontier Sciences, the University of Tokyo, Kashiwanoha 5-1-5, Kashiwa, Chiba277-8561, Japan
| | - Kazune Inatomi
- Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Maidashi 3-1-1, Higashi-Ku, Fukuoka 812-8582, Japan
| | - Yutaka Suzuki
- Life Science Data Research Center, Graduate School of Frontier Sciences, the University of Tokyo, Kashiwanoha 5-1-5, Kashiwa, Chiba277-8561, Japan
| | - Takashi Ito
- Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Maidashi 3-1-1, Higashi-Ku, Fukuoka 812-8582, Japan
| |
Collapse
|
|
2
|
Maji RK, Leisegang MS, Boon RA, Schulz MH. Revealing microRNA regulation in single cells. Trends Genet 2025:S0168-9525(24)00317-2. [PMID: 39863489 DOI: 10.1016/j.tig.2024.12.009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2024] [Revised: 12/22/2024] [Accepted: 12/26/2024] [Indexed: 01/27/2025]
Abstract
MicroRNAs (miRNAs) are key regulators of gene expression and control cellular functions in physiological and pathophysiological states. miRNAs play important roles in disease, stress, and development, and are now being investigated for therapeutic approaches. Alternative processing of miRNAs during biogenesis results in the generation of miRNA isoforms (isomiRs) which further diversify miRNA gene regulation. Single-cell RNA-sequencing (scsRNA-seq) technologies, together with computational strategies, enable exploration of miRNAs, isomiRs, and interacting RNAs at the cellular level. By integration with other miRNA-associated single-cell modalities, miRNA roles can be resolved at different stages of processing and regulation. In this review we discuss (i) single-cell experimental assays that measure miRNA and isomiR abundances, and (ii) computational methods for their analysis to investigate the mechanisms of miRNA biogenesis and post-transcriptional regulation.
Collapse
Affiliation(s)
- Ranjan K Maji
- Institute for Computational Genomic Medicine, Goethe University Frankfurt, Frankfurt, Germany; German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Frankfurt, Germany
| | - Matthias S Leisegang
- German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Frankfurt, Germany; Institute for Cardiovascular Physiology, Goethe University Frankfurt, Frankfurt, Germany
| | - Reinier A Boon
- German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Frankfurt, Germany; Department of Physiology, Amsterdam UMC, VU University, De Boelelaan 1117, 1081, HV, Amsterdam, The Netherlands
| | - Marcel H Schulz
- Institute for Computational Genomic Medicine, Goethe University Frankfurt, Frankfurt, Germany; German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Frankfurt, Germany.
| |
Collapse
|
|
3
|
Xuan DTM, Yeh IJ, Liu HL, Su CY, Ko CC, Ta HDK, Jiang JZ, Sun Z, Lin HY, Wang CY, Yen MC. A comparative analysis of Marburg virus-infected bat and human models from public high-throughput sequencing data. Int J Med Sci 2025; 22:1-16. [PMID: 39744175 PMCID: PMC11659840 DOI: 10.7150/ijms.100696] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/09/2024] [Accepted: 10/10/2024] [Indexed: 02/01/2025] Open
Abstract
Marburg virus (MARV) disease (MVD) is an uncommon yet serious viral hemorrhagic fever that impacts humans and non-human primates. In humans, infection by the MARV is marked by rapid onset, high transmissibility, and elevated mortality rates, presenting considerable obstacles to the development of vaccines and treatments. Bats, particularly Rousettus aegyptiacus, are suspected to be natural hosts of MARV. Previous research reported asymptomatic MARV infection in bats, in stark contrast to the severe responses observed in humans and other primates. Recent MARV outbreaks highlight significant public health concerns, underscoring the need for gene expression studies during MARV progression. To investigate this, we employed two models from the Gene Expression Omnibus, including kidney cells from Rousettus aegyptiacus and primary proximal tubular cells from Homo sapiens. These models were chosen to identify changes in gene expression profiles and to examine co-regulated genes and pathways involved in MARV disease progression. Our analysis of differentially expressed genes (DEGs) revealed that these genes are mainly associated with pathways related to the complement system, innate immune response via interferons (IFNs), Wnt/β-catenin signaling, and Hedgehog signaling, which played crucial roles in MARV infection across both models. Furthermore, we also identified several potential compounds that may be useful against MARV infection. These findings offer valuable insights into the mechanisms underlying MARV's pathophysiology and suggest potential strategies for preventing transmission, managing post-infection effects, and developing future vaccines.
Collapse
Affiliation(s)
- Do Thi Minh Xuan
- Faculty of Pharmacy, Van Lang University, 69/68 Dang Thuy Tram Street, Ward 13, Binh Thanh District, Ho Chi Minh City 70000, Vietnam
| | - I-Jeng Yeh
- Department of Emergency Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
- Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
| | - Hsin-Liang Liu
- Department of Emergency Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
| | - Che-Yu Su
- Department of Emergency Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
| | - Ching-Chung Ko
- Department of Medical Imaging, Chi-Mei Medical Center, Tainan, Taiwan
- Department of Health and Nutrition, Chia Nan University of Pharmacy and Science, Tainan, Taiwan
- School of Medicine, College of Medicine, National Sun Yat-Sen University, Kaohsiung, Taiwan
| | - Hoang Dang Khoa Ta
- Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science, Taipei Medical University, Taipei 11031, Taiwan
| | - Jia-Zhen Jiang
- Emergency Department, Huashan Hospital North, Fudan University, Shanghai 201508, People's Republic of China
| | - Zhengda Sun
- Kaiser Permanente, Northern California Regional Laboratories, The Permanente Medical Group, 1725 Eastshore Hwy, Berkeley, CA 94710, USA
| | - Hung-Yun Lin
- Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan
- Cancer Center, Wan Fang Hospital, Taipei Medical University, Taipei 11031, Taiwan
- TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei 11031, Taiwan
- Traditional Herbal Medicine Research Center of Taipei Medical University Hospital, Taipei Medical University, Taipei 11031, Taiwan
- Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Rensselaer, NY 12144, USA
| | - Chih-Yang Wang
- Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science, Taipei Medical University, Taipei 11031, Taiwan
- Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan
- TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei 11031, Taiwan
| | - Meng-Chi Yen
- Department of Emergency Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
- Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
| |
Collapse
|
|
4
|
Hiers NM, Li L, Li T, Sheng P, Wang Y, Traugot CM, Yao M, Xie M. An endogenous cluster of target-directed microRNA degradation sites induces decay of distinct microRNA families. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.12.11.627053. [PMID: 39713366 PMCID: PMC11661237 DOI: 10.1101/2024.12.11.627053] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 12/24/2024]
Abstract
While much is known about miRNA biogenesis and canonical miRNA targeting, relatively less is understood about miRNA decay. The major miRNA decay pathway in metazoans is mediated through target-directed miRNA degradation (TDMD), in which certain RNAs can "trigger" miRNA decay. All known triggers for TDMD base pair with the miRNA seed, and extensively base pair on the miRNA 3' end, a pattern that supposedly induces a TDMD-competent conformational change of Argonaute (Ago), allowing for miRNA turnover. Here, we utilized Ago1-CLASH to find that the Drosophila transcript Kah contains at least two triggers, a "trigger cluster", against miR-9b and the miR-279 family. One of these triggers contains minimal/non-canonical 3' end base pairing but is still sufficient to induce TDMD of the entire miR-279 family. We found that these clustered triggers likely lack cooperativity, the minimal 3' pairing is required for miR-279 family turnover, and probed the in-cell RNA structure of the Kah trigger cluster. Overall, this study expands the list of endogenous triggers and the unexpectedly complex regulatory network governing miRNA degradation.
Collapse
Affiliation(s)
- Nicholas M. Hiers
- Department of Biochemistry and Molecular Biology, University of
Florida, Gainesville, FL, 32610, USA
- UF Health Cancer Center, University of Florida, Gainesville, FL,
32610, USA
| | - Lu Li
- Department of Biochemistry and Molecular Biology, University of
Florida, Gainesville, FL, 32610, USA
- UF Health Cancer Center, University of Florida, Gainesville, FL,
32610, USA
| | - Tianqi Li
- Department of Biochemistry and Molecular Biology, University of
Florida, Gainesville, FL, 32610, USA
- UF Health Cancer Center, University of Florida, Gainesville, FL,
32610, USA
| | - Peike Sheng
- Department of Biochemistry and Molecular Biology, University of
Florida, Gainesville, FL, 32610, USA
- UF Health Cancer Center, University of Florida, Gainesville, FL,
32610, USA
| | - Yuzhi Wang
- Department of Biochemistry and Molecular Biology, University of
Florida, Gainesville, FL, 32610, USA
- UF Health Cancer Center, University of Florida, Gainesville, FL,
32610, USA
| | - Conner M. Traugot
- Department of Biochemistry and Molecular Biology, University of
Florida, Gainesville, FL, 32610, USA
- UF Genetics Institute, University of Florida, Gainesville, FL,
32610, USA
| | - Michael Yao
- Department of Biochemistry and Molecular Biology, University of
Florida, Gainesville, FL, 32610, USA
| | - Mingyi Xie
- Department of Biochemistry and Molecular Biology, University of
Florida, Gainesville, FL, 32610, USA
- UF Health Cancer Center, University of Florida, Gainesville, FL,
32610, USA
- UF Genetics Institute, University of Florida, Gainesville, FL,
32610, USA
| |
Collapse
|
|
5
|
Oesinghaus L, Castillo-Hair S, Ludwig N, Keller A, Seelig G. Quantitative design of cell type-specific mRNA stability from microRNA expression data. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.10.28.620728. [PMID: 39554011 PMCID: PMC11565874 DOI: 10.1101/2024.10.28.620728] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 11/19/2024]
Abstract
Limiting expression to target cell types is a longstanding goal in gene therapy, which could be met by sensing endogenous microRNA. However, an unclear association between microRNA expression and activity currently hampers such an approach. Here, we probe this relationship by measuring the stability of synthetic microRNA-responsive 3'UTRs across 10 cell lines in a library format. By systematically addressing biases in microRNA expression data and confounding factors such as microRNA crosstalk, we demonstrate that a straightforward model can quantitatively predict reporter stability purely from expression data. We use this model to design constructs with previously unattainable response patterns across our cell lines. The rules we derive for microRNA expression data selection and processing should apply to microRNA- responsive devices for any environment with available expression data.
Collapse
|
|
6
|
Booy EP, Gussakovsky D, Brown M, Shwaluk R, Nachtigal MW, McKenna SA. lncRNA BC200 is processed into a stable Alu monomer. RNA (NEW YORK, N.Y.) 2024; 30:1477-1494. [PMID: 39179355 PMCID: PMC11482611 DOI: 10.1261/rna.080152.124] [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: 06/21/2024] [Accepted: 08/08/2024] [Indexed: 08/26/2024]
Abstract
The noncoding RNA BC200 is elevated in human cancers and is implicated in translation regulation as well as cell survival and proliferation. Upon BC200 overexpression, we observed correlated expression of a second, smaller RNA species. This RNA is expressed endogenously and exhibits cell-type-dependent variability relative to BC200. Aptamer-tagged expression constructs confirmed that the RNA is a truncated form of BC200, and sequencing revealed a modal length of 120 nt; thus, we refer to the RNA fragment as BC120. We present a methodology for accurate and specific detection of BC120 and establish that BC120 is expressed in several normal human tissues and is also elevated in ovarian cancer. BC120 exhibits remarkable stability relative to BC200 and is resistant to knockdown strategies that target the 3' unique sequence of BC200. Combined knockdown of BC200 and BC120 exhibits greater phenotypic impacts than knockdown of BC200 alone, and overexpression of BC120 negatively impacts translation of a GFP reporter, providing insight into a potential translational regulatory role for this RNA. The presence of a novel, truncated, and stable form of BC200 adds complexity to the investigation of this noncoding RNA that must be considered in future studies of BC200 and other related Alu RNAs.
Collapse
Affiliation(s)
- Evan P Booy
- Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
| | - Daniel Gussakovsky
- Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
| | - Mira Brown
- Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
| | - Rowan Shwaluk
- Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
| | - Mark W Nachtigal
- Department of Biochemistry and Medical Genetics, Gynecology and Reproductive Sciences, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0J9
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0J9
- Paul Albrechtsen Research Institute, CancerCare Manitoba, Winnipeg, Manitoba, Canada R2H 2A6
| | - Sean A McKenna
- Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
| |
Collapse
|
|
7
|
Rappol T, Waldl M, Chugunova A, Hofacker I, Pauli A, Vilardo E. tRNA expression and modification landscapes, and their dynamics during zebrafish embryo development. Nucleic Acids Res 2024; 52:10575-10594. [PMID: 38989621 PMCID: PMC11417395 DOI: 10.1093/nar/gkae595] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2024] [Revised: 06/19/2024] [Accepted: 06/26/2024] [Indexed: 07/12/2024] Open
Abstract
tRNA genes exist in multiple copies in the genome of all organisms across the three domains of life. Besides the sequence differences across tRNA copies, extensive post-transcriptional modification adds a further layer to tRNA diversification. Whilst the crucial role of tRNAs as adapter molecules in protein translation is well established, whether all tRNAs are actually expressed, and whether the differences across isodecoders play any regulatory role is only recently being uncovered. Here we built upon recent developments in the use of NGS-based methods for RNA modification detection and developed tRAM-seq, an experimental protocol and in silico analysis pipeline to investigate tRNA expression and modification. Using tRAM-seq, we analysed the full ensemble of nucleo-cytoplasmic and mitochondrial tRNAs during embryonic development of the model vertebrate zebrafish. We show that the repertoire of tRNAs changes during development, with an apparent major switch in tRNA isodecoder expression and modification profile taking place around the start of gastrulation. Taken together, our findings suggest the existence of a general reprogramming of the expressed tRNA pool, possibly gearing the translational machinery for distinct stages of the delicate and crucial process of embryo development.
Collapse
Affiliation(s)
- Tom Rappol
- Center for Anatomy & Cell Biology, Medical University of Vienna, 1090 Vienna, Austria
| | - Maria Waldl
- Center for Anatomy & Cell Biology, Medical University of Vienna, 1090 Vienna, Austria
- Department of Theoretical Chemistry, University of Vienna, 1090 Vienna, Austria
- Vienna Doctoral School in Chemistry (DoSChem), University of Vienna, 1090 Vienna, Austria
- Institute of Computer Science and Interdisciplinary Center for Bioinformatics, Leipzig University, D-04107 Leipzig, Germany
| | - Anastasia Chugunova
- Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), 1030 Vienna, Austria
| | - Ivo L Hofacker
- Department of Theoretical Chemistry, University of Vienna, 1090 Vienna, Austria
- Faculty of Computer Science, Research Group Bioinformatics and Computational Biology, University of Vienna, 1090 Vienna, Austria
| | - Andrea Pauli
- Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), 1030 Vienna, Austria
| | - Elisa Vilardo
- Center for Anatomy & Cell Biology, Medical University of Vienna, 1090 Vienna, Austria
| |
Collapse
|
|
8
|
Jusic A, Erpapazoglou Z, Dalgaard LT, Lakkisto P, de Gonzalo-Calvo D, Benczik B, Ágg B, Ferdinandy P, Fiedorowicz K, Schroen B, Lazou A, Devaux Y, on behalf of EU-CardioRNA COST Action CA17129, AtheroNET COST Action CA21153. Guidelines for mitochondrial RNA analysis. MOLECULAR THERAPY. NUCLEIC ACIDS 2024; 35:102262. [PMID: 39091381 PMCID: PMC11292373 DOI: 10.1016/j.omtn.2024.102262] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Indexed: 08/04/2024]
Abstract
Mitochondria are the energy-producing organelles of mammalian cells with critical involvement in metabolism and signaling. Studying their regulation in pathological conditions may lead to the discovery of novel drugs to treat, for instance, cardiovascular or neurological diseases, which affect high-energy-consuming cells such as cardiomyocytes, hepatocytes, or neurons. Mitochondria possess both protein-coding and noncoding RNAs, such as microRNAs, long noncoding RNAs, circular RNAs, and piwi-interacting RNAs, encoded by the mitochondria or the nuclear genome. Mitochondrial RNAs are involved in anterograde-retrograde communication between the nucleus and mitochondria and play an important role in physiological and pathological conditions. Despite accumulating evidence on the presence and biogenesis of mitochondrial RNAs, their study continues to pose significant challenges. Currently, there are no standardized protocols and guidelines to conduct deep functional characterization and expression profiling of mitochondrial RNAs. To overcome major obstacles in this emerging field, the EU-CardioRNA and AtheroNET COST Action networks summarize currently available techniques and emphasize critical points that may constitute sources of variability and explain discrepancies between published results. Standardized methods and adherence to guidelines to quantify and study mitochondrial RNAs in normal and disease states will improve research outputs, their reproducibility, and translation potential to clinical application.
Collapse
Affiliation(s)
- Amela Jusic
- HAYA Therapeutics SA, Route De La Corniche 6, SuperLab Suisse - Batiment Serine, 1066 Epalinges, Switzerland
- Cardiovascular Research Unit, Department of Precision Health, Luxembourg Institute of Health, 1445 Strassen, Luxembourg
| | - Zoi Erpapazoglou
- Ιnstitute for Fundamental Biomedical Research, B.S.R.C. “Alexander Fleming”, Vari, 16672 Athens, Greece
| | - Louise Torp Dalgaard
- Department of Science and Environment, Roskilde University, 4000 Roskilde, Denmark
| | - Päivi Lakkisto
- Minerva Foundation Institute for Medical Research, 00290 Helsinki, Finland
- Department of Clinical Chemistry, University of Helsinki and Helsinki University Hospital, 00014 Helsinki, Finland
| | - David de Gonzalo-Calvo
- Translational Research in Respiratory Medicine, University Hospital Arnau de Vilanova and Santa Maria, IRBLleida, 25198 Lleida, Spain
- CIBER of Respiratory Diseases (CIBERES), Institute of Health Carlos III, 28029 Madrid, Spain
| | - Bettina Benczik
- Cardiometabolic and HUN-REN-SU System Pharmacology Research Group, Center for Pharmacology and Drug Research & Development, Department of Pharmacology and Pharmacotherapy, Semmelweis University, 1089 Budapest, Hungary
- Pharmahungary Group, 6722 Szeged, Hungary
| | - Bence Ágg
- Cardiometabolic and HUN-REN-SU System Pharmacology Research Group, Center for Pharmacology and Drug Research & Development, Department of Pharmacology and Pharmacotherapy, Semmelweis University, 1089 Budapest, Hungary
- Pharmahungary Group, 6722 Szeged, Hungary
| | - Péter Ferdinandy
- Cardiometabolic and HUN-REN-SU System Pharmacology Research Group, Center for Pharmacology and Drug Research & Development, Department of Pharmacology and Pharmacotherapy, Semmelweis University, 1089 Budapest, Hungary
- Pharmahungary Group, 6722 Szeged, Hungary
| | | | - Blanche Schroen
- Department of Physiology, Cardiovascular Research Institute Maastricht, Maastricht University, ER 6229 Maastricht, the Netherlands
| | - Antigone Lazou
- School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
| | - Yvan Devaux
- Cardiovascular Research Unit, Department of Precision Health, Luxembourg Institute of Health, 1445 Strassen, Luxembourg
| | - on behalf of EU-CardioRNA COST Action CA17129
- HAYA Therapeutics SA, Route De La Corniche 6, SuperLab Suisse - Batiment Serine, 1066 Epalinges, Switzerland
- Cardiovascular Research Unit, Department of Precision Health, Luxembourg Institute of Health, 1445 Strassen, Luxembourg
- Ιnstitute for Fundamental Biomedical Research, B.S.R.C. “Alexander Fleming”, Vari, 16672 Athens, Greece
- Department of Science and Environment, Roskilde University, 4000 Roskilde, Denmark
- Minerva Foundation Institute for Medical Research, 00290 Helsinki, Finland
- Department of Clinical Chemistry, University of Helsinki and Helsinki University Hospital, 00014 Helsinki, Finland
- Translational Research in Respiratory Medicine, University Hospital Arnau de Vilanova and Santa Maria, IRBLleida, 25198 Lleida, Spain
- CIBER of Respiratory Diseases (CIBERES), Institute of Health Carlos III, 28029 Madrid, Spain
- Cardiometabolic and HUN-REN-SU System Pharmacology Research Group, Center for Pharmacology and Drug Research & Development, Department of Pharmacology and Pharmacotherapy, Semmelweis University, 1089 Budapest, Hungary
- Pharmahungary Group, 6722 Szeged, Hungary
- NanoBioMedical Centre, Adam Mickiewicz University in Poznan, 61614 Poznan, Poland
- Department of Physiology, Cardiovascular Research Institute Maastricht, Maastricht University, ER 6229 Maastricht, the Netherlands
- School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
| | - AtheroNET COST Action CA21153
- HAYA Therapeutics SA, Route De La Corniche 6, SuperLab Suisse - Batiment Serine, 1066 Epalinges, Switzerland
- Cardiovascular Research Unit, Department of Precision Health, Luxembourg Institute of Health, 1445 Strassen, Luxembourg
- Ιnstitute for Fundamental Biomedical Research, B.S.R.C. “Alexander Fleming”, Vari, 16672 Athens, Greece
- Department of Science and Environment, Roskilde University, 4000 Roskilde, Denmark
- Minerva Foundation Institute for Medical Research, 00290 Helsinki, Finland
- Department of Clinical Chemistry, University of Helsinki and Helsinki University Hospital, 00014 Helsinki, Finland
- Translational Research in Respiratory Medicine, University Hospital Arnau de Vilanova and Santa Maria, IRBLleida, 25198 Lleida, Spain
- CIBER of Respiratory Diseases (CIBERES), Institute of Health Carlos III, 28029 Madrid, Spain
- Cardiometabolic and HUN-REN-SU System Pharmacology Research Group, Center for Pharmacology and Drug Research & Development, Department of Pharmacology and Pharmacotherapy, Semmelweis University, 1089 Budapest, Hungary
- Pharmahungary Group, 6722 Szeged, Hungary
- NanoBioMedical Centre, Adam Mickiewicz University in Poznan, 61614 Poznan, Poland
- Department of Physiology, Cardiovascular Research Institute Maastricht, Maastricht University, ER 6229 Maastricht, the Netherlands
- School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
| |
Collapse
|
|
9
|
Davidsen K, Sullivan LB. A robust method for measuring aminoacylation through tRNA-Seq. eLife 2024; 12:RP91554. [PMID: 39076160 PMCID: PMC11288633 DOI: 10.7554/elife.91554] [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] [Indexed: 07/31/2024] Open
Abstract
Current methods to quantify the fraction of aminoacylated tRNAs, also known as the tRNA charge, are limited by issues with either low throughput, precision, and/or accuracy. Here, we present an optimized charge transfer RNA sequencing (tRNA-Seq) method that combines previous developments with newly described approaches to establish a protocol for precise and accurate tRNA charge measurements. We verify that this protocol provides robust quantification of tRNA aminoacylation and we provide an end-to-end method that scales to hundreds of samples including software for data processing. Additionally, we show that this method supports measurements of relative tRNA expression levels and can be used to infer tRNA modifications through reverse transcription misincorporations, thereby supporting multipurpose applications in tRNA biology.
Collapse
Affiliation(s)
- Kristian Davidsen
- Human Biology Division, Fred Hutchinson Cancer Research CenterSeattleUnited States
- Molecular and Cellular Biology Program, University of WashingtonSeattleUnited States
| | - Lucas B Sullivan
- Human Biology Division, Fred Hutchinson Cancer Research CenterSeattleUnited States
| |
Collapse
|
|
10
|
Fernandez MV, Liu M, Beric A, Johnson M, Cetin A, Patel M, Budde J, Kohlfeld P, Bergmann K, Lowery J, Flynn A, Brock W, Sanchez Montejo B, Gentsch J, Sykora N, Norton J, Gentsch J, Valdez O, Gorijala P, Sanford J, Sun Y, Wang C, Western D, Timsina J, Mangetti Goncalves T, Do AN, Sung YJ, Zhao G, Morris JC, Moulder K, Holtzman DM, Bateman RJ, Karch C, Hassenstab J, Xiong C, Schindler SE, Balls-Berry JJ, Benzinger TLS, Perrin RJ, Denny A, Snider BJ, Stark SL, Ibanez L, Cruchaga C. Genetic and multi-omic resources for Alzheimer disease and related dementia from the Knight Alzheimer Disease Research Center. Sci Data 2024; 11:768. [PMID: 38997326 PMCID: PMC11245521 DOI: 10.1038/s41597-024-03485-9] [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: 01/24/2024] [Accepted: 06/06/2024] [Indexed: 07/14/2024] Open
Abstract
The Knight-Alzheimer Disease Research Center (Knight-ADRC) at Washington University in St. Louis has pioneered and led worldwide seminal studies that have expanded our clinical, social, pathological, and molecular understanding of Alzheimer Disease. Over more than 40 years, research volunteers have been recruited to participate in cognitive, neuropsychologic, imaging, fluid biomarkers, genomic and multi-omic studies. Tissue and longitudinal data collected to foster, facilitate, and support research on dementia and aging. The Genetics and high throughput -omics core (GHTO) have collected of more than 26,000 biological samples from 6,625 Knight-ADRC participants. Samples available include longitudinal DNA, RNA, non-fasted plasma, cerebrospinal fluid pellets, and peripheral blood mononuclear cells. The GHTO has performed deep molecular profiling (genomic, transcriptomic, epigenomic, proteomic, and metabolomic) from large number of brain (n = 2,117), CSF (n = 2,012) and blood/plasma (n = 8,265) samples with the goal of identifying novel risk and protective variants, identify novel molecular biomarkers and causal and druggable targets. Overall, the resources available at GHTO support the increase of our understanding of Alzheimer Disease.
Collapse
Affiliation(s)
- Maria Victoria Fernandez
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
- Research Center and Memory Clinic, ACE Alzheimer Center, Barcelona, Spain
| | - Menghan Liu
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Aleksandra Beric
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Matt Johnson
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Arda Cetin
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Maulik Patel
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - John Budde
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Pat Kohlfeld
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Kristy Bergmann
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Joseph Lowery
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Allison Flynn
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - William Brock
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Brenda Sanchez Montejo
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Jen Gentsch
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Nicholas Sykora
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Joanne Norton
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Jen Gentsch
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Olga Valdez
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Priyanka Gorijala
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Jessie Sanford
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Yichen Sun
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Ciyang Wang
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Dan Western
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Jigyasha Timsina
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | | | - Anh N Do
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
- Division of Biostatistics, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Yun Ju Sung
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Guoyan Zhao
- Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA
- Department of Neurology, Washington University School of Medicine, St. Louis, MO, 63110, USA
- Pathology and Immunology Department, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - John C Morris
- Department of Neurology, Washington University School of Medicine, St. Louis, MO, 63110, USA
- Knight Alzheimer Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA
| | - Krista Moulder
- Department of Neurology, Washington University School of Medicine, St. Louis, MO, 63110, USA
- Knight Alzheimer Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA
| | - David M Holtzman
- Department of Neurology, Washington University School of Medicine, St. Louis, MO, 63110, USA
- Knight Alzheimer Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA
- Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA
| | - Randall J Bateman
- Department of Neurology, Washington University School of Medicine, St. Louis, MO, 63110, USA
- Knight Alzheimer Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA
- Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA
- Dominantly Inherited Alzheimer Disease Network (DIAN), St. Louis, USA
| | - Celeste Karch
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA
- Knight Alzheimer Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA
- Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA
- Dominantly Inherited Alzheimer Disease Network (DIAN), St. Louis, USA
| | - Jason Hassenstab
- Department of Neurology, Washington University School of Medicine, St. Louis, MO, 63110, USA
- Knight Alzheimer Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA
| | - Chengjie Xiong
- Division of Biostatistics, Washington University School of Medicine, St. Louis, MO, 63110, USA
- Department of Neurology, Washington University School of Medicine, St. Louis, MO, 63110, USA
- Knight Alzheimer Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA
- Dominantly Inherited Alzheimer Disease Network (DIAN), St. Louis, USA
| | - Suzanne E Schindler
- Department of Neurology, Washington University School of Medicine, St. Louis, MO, 63110, USA
- Knight Alzheimer Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA
| | - Joyce Joy Balls-Berry
- Department of Neurology, Washington University School of Medicine, St. Louis, MO, 63110, USA
- Knight Alzheimer Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA
| | - Tammie L S Benzinger
- Department of Neurology, Washington University School of Medicine, St. Louis, MO, 63110, USA
- Knight Alzheimer Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA
- Dominantly Inherited Alzheimer Disease Network (DIAN), St. Louis, USA
- Radiology Department, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Richard J Perrin
- Pathology and Immunology Department, Washington University School of Medicine, St. Louis, MO, 63110, USA
- Knight Alzheimer Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA
- Dominantly Inherited Alzheimer Disease Network (DIAN), St. Louis, USA
| | - Andrea Denny
- Department of Neurology, Washington University School of Medicine, St. Louis, MO, 63110, USA
- Knight Alzheimer Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA
| | - B Joy Snider
- Department of Neurology, Washington University School of Medicine, St. Louis, MO, 63110, USA
- Knight Alzheimer Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA
- Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA
| | - Susan L Stark
- Knight Alzheimer Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA
- Occupational Therapy, Neurology and Social Work, St. Louis, USA
| | - Laura Ibanez
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA.
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA.
- Department of Neurology, Washington University School of Medicine, St. Louis, MO, 63110, USA.
- Dominantly Inherited Alzheimer Disease Network (DIAN), St. Louis, USA.
| | - Carlos Cruchaga
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA.
- NeuroGenomics and Informatics Center, Washington University School of Medicine, St. Louis, MO, 63110, USA.
- Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA.
- Department of Neurology, Washington University School of Medicine, St. Louis, MO, 63110, USA.
- Knight Alzheimer Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA.
- Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA.
- Dominantly Inherited Alzheimer Disease Network (DIAN), St. Louis, USA.
| |
Collapse
|
|
11
|
Yuan Y, DeMott MS, Byrne SR, Global Microbiome Conservancy, Dedon PC. PT-seq: A method for metagenomic analysis of phosphorothioate epigenetics in complex microbial communities. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.06.03.597111. [PMID: 38895297 PMCID: PMC11185561 DOI: 10.1101/2024.06.03.597111] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/21/2024]
Abstract
Among dozens of known epigenetic marks, naturally occurring phosphorothioate (PT) DNA modifications are unique in replacing a non-bridging phosphate oxygen with redox-active sulfur and function in prokaryotic restriction-modification and transcriptional regulation. Interest in PTs has grown due to the widespread distribution of the dnd, ssp, and brx genes among bacteria and archaea, as well as the discovery of PTs in 5-10% of gut microbes. Efforts to map PTs in complex microbiomes using existing next-generation and direct sequencing technologies have failed due to poor sensitivity. Here we developed PT-seq as a high-sensitivity method to quantitatively map PTs across genomes and metagenomically identify PT-containing microbes in complex genomic mixtures. Like other methods for mapping PTs in individual genomes, PT-seq exploits targeted DNA strand cleavage at PTs by iodine, followed by sequencing library construction using ligation or template switching approaches. However, PT-specific sequencing reads are dramatically increased by adding steps to heat denature the DNA, block pre-existing 3'-ends, fragment DNA after T-tailing, and enrich iodine-induced breaks using biotin-labeling and streptavidin beads capture. Iterative optimization of the sensitivity and specificity of PT-seq is demonstrated with individual bacteria and human fecal DNA.
Collapse
Affiliation(s)
- Yifeng Yuan
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
| | - Michael S. DeMott
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
- Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
| | - Shane R. Byrne
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
| | | | - Peter C. Dedon
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
- Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
- Antimicrobial Resistance IRG, Singapore-MIT Alliance for Research and Technology, Singapore
| |
Collapse
|
|
12
|
Rousseau M, Oulavallickal T, Williamson A, Arcus V, Patrick WM, Hicks J. Characterisation and engineering of a thermophilic RNA ligase from Palaeococcus pacificus. Nucleic Acids Res 2024; 52:3924-3937. [PMID: 38421610 DOI: 10.1093/nar/gkae149] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2023] [Revised: 01/23/2024] [Accepted: 02/19/2024] [Indexed: 03/02/2024] Open
Abstract
RNA ligases are important enzymes in molecular biology and are highly useful for the manipulation and analysis of nucleic acids, including adapter ligation in next-generation sequencing of microRNAs. Thermophilic RNA ligases belonging to the RNA ligase 3 family are gaining attention for their use in molecular biology, for example a thermophilic RNA ligase from Methanobacterium thermoautotrophicum is commercially available for the adenylation of nucleic acids. Here we extensively characterise a newly identified RNA ligase from the thermophilic archaeon Palaeococcus pacificus (PpaRnl). PpaRnl exhibited significant substrate adenylation activity but low ligation activity across a range of oligonucleotide substrates. Mutation of Lys92 in motif I to alanine, resulted in an enzyme that lacked adenylation activity, but demonstrated improved ligation activity with pre-adenylated substrates (ATP-independent ligation). Subsequent structural characterisation revealed that in this mutant enzyme Lys238 was found in two alternate positions for coordination of the phosphate tail of ATP. In contrast mutation of Lys238 in motif V to glycine via structure-guided engineering enhanced ATP-dependent ligation activity via an arginine residue compensating for the absence of Lys238. Ligation activity for both mutations was higher than the wild-type, with activity observed across a range of oligonucleotide substrates with varying sequence and secondary structure.
Collapse
Affiliation(s)
- Meghan Rousseau
- School of Science, The University of Waikato, Hamilton 3216, New Zealand
| | - Tifany Oulavallickal
- School of Biological Sciences, Victoria University of Wellington, Wellington 6140, New Zealand
| | - Adele Williamson
- School of Science, The University of Waikato, Hamilton 3216, New Zealand
| | - Vic Arcus
- School of Science, The University of Waikato, Hamilton 3216, New Zealand
| | - Wayne M Patrick
- School of Biological Sciences, Victoria University of Wellington, Wellington 6140, New Zealand
| | - Joanna Hicks
- Te Huataki Waiora School of Health, The University of Waikato, Hamilton 3216, New Zealand
| |
Collapse
|
|
13
|
Davidsen K, Sullivan LB. A robust method for measuring aminoacylation through tRNA-Seq. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2023.07.31.551363. [PMID: 37577502 PMCID: PMC10418082 DOI: 10.1101/2023.07.31.551363] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/15/2023]
Abstract
Current methods to quantify the fraction of aminoacylated tRNAs, also known as the tRNA charge, are limited by issues with either low throughput, precision, and/or accuracy. Here, we present an optimized charge tRNA-Seq method that combines previous developments with newly described approaches to establish a protocol for precise and accurate tRNA charge measurements. We verify that this protocol provides robust quantification of tRNA aminoacylation and we provide an end-to-end method that scales to hundreds of samples including software for data processing. Additionally, we show that this method supports measurements of relative tRNA expression levels and can be used to infer tRNA modifications through reverse transcription misincorporations, thereby supporting multipurpose applications in tRNA biology.
Collapse
Affiliation(s)
- Kristian Davidsen
- Human Biology Division, Fred Hutchinson Cancer Center, United States
- Molecular and Cellular Biology Program, University of Washington, United States
| | - Lucas B Sullivan
- Human Biology Division, Fred Hutchinson Cancer Center, United States
| |
Collapse
|
|
14
|
Hiers NM, Li T, Traugot CM, Xie M. Target-directed microRNA degradation: Mechanisms, significance, and functional implications. WILEY INTERDISCIPLINARY REVIEWS. RNA 2024; 15:e1832. [PMID: 38448799 PMCID: PMC11098282 DOI: 10.1002/wrna.1832] [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: 12/08/2023] [Revised: 01/11/2024] [Accepted: 01/12/2024] [Indexed: 03/08/2024]
Abstract
MicroRNAs (miRNAs) are small non-coding RNAs that play a fundamental role in enabling miRNA-mediated target repression, a post-transcriptional gene regulatory mechanism preserved across metazoans. Loss of certain animal miRNA genes can lead to developmental abnormalities, disease, and various degrees of embryonic lethality. These short RNAs normally guide Argonaute (AGO) proteins to target RNAs, which are in turn translationally repressed and destabilized, silencing the target to fine-tune gene expression and maintain cellular homeostasis. Delineating miRNA-mediated target decay has been thoroughly examined in thousands of studies, yet despite these exhaustive studies, comparatively less is known about how and why miRNAs are directed for decay. Several key observations over the years have noted instances of rapid miRNA turnover, suggesting endogenous means for animals to induce miRNA degradation. Recently, it was revealed that certain targets, so-called target-directed miRNA degradation (TDMD) triggers, can "trigger" miRNA decay through inducing proteolysis of AGO and thereby the bound miRNA. This process is mediated in animals via the ZSWIM8 ubiquitin ligase complex, which is recruited to AGO during engagement with triggers. Since its discovery, several studies have identified that ZSWIM8 and TDMD are indispensable for proper animal development. Given the rapid expansion of this field of study, here, we summarize the key findings that have led to and followed the discovery of ZSWIM8-dependent TDMD. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA in Disease and Development > RNA in Development.
Collapse
Affiliation(s)
- Nicholas M Hiers
- Department of Biochemistry and Molecular Biology, College of Medicine, UF Health Cancer Center, UF Genetics Institute, University of Florida, Gainesville, Florida, USA
| | - Tianqi Li
- Department of Biochemistry and Molecular Biology, College of Medicine, UF Health Cancer Center, UF Genetics Institute, University of Florida, Gainesville, Florida, USA
| | - Conner M Traugot
- Department of Biochemistry and Molecular Biology, College of Medicine, UF Health Cancer Center, UF Genetics Institute, University of Florida, Gainesville, Florida, USA
| | - Mingyi Xie
- Department of Biochemistry and Molecular Biology, College of Medicine, UF Health Cancer Center, UF Genetics Institute, University of Florida, Gainesville, Florida, USA
| |
Collapse
|
|
15
|
Busseau I, Mockly S, Houbron É, Somaï H, Seitz H. Evaluation of microRNA variant maturation prior to genome edition. Biochimie 2024; 217:86-94. [PMID: 37385398 DOI: 10.1016/j.biochi.2023.06.007] [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: 12/16/2022] [Revised: 05/22/2023] [Accepted: 06/13/2023] [Indexed: 07/01/2023]
Abstract
Assessment of the functionality of individual microRNA/target sites is a crucial issue. Genome editing techniques should theoretically permit a fine functional exploration of such interactions, allowing the mutation of microRNAs or individual binding sites in a complete in vivo setting, therefore abrogating or restoring individual interactions on demand. A major limitation to this experimental strategy is the influence of microRNA sequence on its accumulation level, which introduces a confounding effect when assessing phenotypic rescue by compensatorily mutated microRNA and target site. Here we describe a simple assay to identify microRNA variants most likely to accumulate at wild-type levels even though their sequence has been mutated. In this assay, quantification of a reporter construct in cultured cells predicts the efficiency of an early biogenesis step, the Drosha-dependent cleavage of microRNA precursors, which appears to be a major determinant of microRNA accumulation in our variant collection. This system allowed the generation of a mutant Drosophila strain expressing a bantam microRNA variant at wild-type levels.
Collapse
Affiliation(s)
- Isabelle Busseau
- Institut de Génétique Humaine, UMR 9002, CNRS and University of Montpellier, Montpellier, France.
| | - Sophie Mockly
- Institut de Génétique Humaine, UMR 9002, CNRS and University of Montpellier, Montpellier, France
| | - Élisabeth Houbron
- Institut de Génétique Humaine, UMR 9002, CNRS and University of Montpellier, Montpellier, France
| | - Hedi Somaï
- Institut de Génétique Humaine, UMR 9002, CNRS and University of Montpellier, Montpellier, France
| | - Hervé Seitz
- Institut de Génétique Humaine, UMR 9002, CNRS and University of Montpellier, Montpellier, France.
| |
Collapse
|
|
16
|
Seki M, Kuze Y, Zhang X, Kurotani KI, Notaguchi M, Nishio H, Kudoh H, Suzaki T, Yoshida S, Sugano S, Matsushita T, Suzuki Y. An improved method for the highly specific detection of transcription start sites. Nucleic Acids Res 2024; 52:e7. [PMID: 37994784 PMCID: PMC10810191 DOI: 10.1093/nar/gkad1116] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Revised: 10/17/2023] [Accepted: 11/06/2023] [Indexed: 11/24/2023] Open
Abstract
Precise detection of the transcriptional start site (TSS) is a key for characterizing transcriptional regulation of genes and for annotation of newly sequenced genomes. Here, we describe the development of an improved method, designated 'TSS-seq2.' This method is an iterative improvement of TSS-seq, a previously published enzymatic cap-structure conversion method to detect TSSs in base sequences. By modifying the original procedure, including by introducing split ligation at the key cap-selection step, the yield and the accuracy of the reaction has been substantially improved. For example, TSS-seq2 can be conducted using as little as 5 ng of total RNA with an overall accuracy of 96%; this yield a less-biased and more precise detection of TSS. We then applied TSS-seq2 for TSS analysis of four plant species that had not yet been analyzed by any previous TSS method.
Collapse
Affiliation(s)
- Masahide Seki
- Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan
| | - Yuta Kuze
- Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan
| | - Xiang Zhang
- Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, Nara, Japan
| | - Ken-ichi Kurotani
- Bioscience and Biotechnology Center, Nagoya University, Aichi, Japan
| | - Michitaka Notaguchi
- Bioscience and Biotechnology Center, Nagoya University, Aichi, Japan
- Department of Botany, Graduate School of Science, Kyoto University, Kyoto, Japan
- Graduate School of Bioagricultural Sciences, Nagoya University, Aichi, Nagoya, Japan
| | - Haruki Nishio
- Data Science and AI Innovation Research Promotion Center, Shiga University, Shiga, Japan
| | - Hiroshi Kudoh
- Center for Ecological Research, Kyoto University, Shiga, Japan
| | - Takuya Suzaki
- Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan
- Tsukuba Plant-Innovation Research Center, University of Tsukuba, Ibaraki, Japan
| | - Satoko Yoshida
- Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, Nara, Japan
| | - Sumio Sugano
- Institute of Kashiwa-no-ha Omics Gate, Chiba, Japan
- Future Medicine Education and Research Organization, Chiba University, Chiba, Japan
| | - Tomonao Matsushita
- Department of Botany, Graduate School of Science, Kyoto University, Kyoto, Japan
| | - Yutaka Suzuki
- Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan
| |
Collapse
|
|
17
|
Wang J, Huang J, Hu Y, Guo Q, Zhang S, Tian J, Niu Y, Ji L, Xu Y, Tang P, He Y, Wang Y, Zhang S, Yang H, Kang K, Chen X, Li X, Yang M, Gou D. Terminal modifications independent cell-free RNA sequencing enables sensitive early cancer detection and classification. Nat Commun 2024; 15:156. [PMID: 38168054 PMCID: PMC10761679 DOI: 10.1038/s41467-023-44461-y] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2023] [Accepted: 12/14/2023] [Indexed: 01/05/2024] Open
Abstract
Cell-free RNAs (cfRNAs) offer an opportunity to detect diseases from a transcriptomic perspective, however, existing techniques have fallen short in generating a comprehensive cell-free transcriptome profile. We develop a sensitive library preparation method that is robust down to 100 µl input plasma to analyze cfRNAs independent of their 5'-end modifications. We show that it outperforms adapter ligation-based method in detecting a greater number of cfRNA species. We perform transcriptome-wide characterizations in 165 lung cancer, 30 breast cancer, 37 colorectal cancer, 55 gastric cancer, 15 liver cancer, and 133 cancer-free participants and demonstrate its ability to identify transcriptomic changes occurring in early-stage tumors. We also leverage machine learning analyses on the differentially expressed cfRNA signatures and reveal their robust performance in cancer detection and classification. Our work sets the stage for in-depth study of the cfRNA repertoire and highlights the value of cfRNAs as cancer biomarkers in clinical applications.
Collapse
Affiliation(s)
- Jun Wang
- College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, Guangdong, China
| | - Jinyong Huang
- College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, Guangdong, China
- College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen, Guangdong, China
| | - Yunlong Hu
- Department of Clinical Laboratory, Peking University Shenzhen Hospital, Shenzhen, Guangdong, China
| | - Qianwen Guo
- College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, Guangdong, China
| | - Shasha Zhang
- College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, Guangdong, China
| | - Jinglin Tian
- College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, Guangdong, China
| | - Yanqin Niu
- College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, Guangdong, China
| | - Ling Ji
- Department of Clinical Laboratory, Peking University Shenzhen Hospital, Shenzhen, Guangdong, China
| | - Yuzhong Xu
- Department of Clinical Laboratory, People's Hospital of Bao'an Shenzhen, Shenzhen, Guangdong, China
| | - Peijun Tang
- Department of Tuberculosis, The Fifth People's Hospital of Suzhou, Suzhou, Jiangsu, China
| | - Yaqin He
- Surgical Department, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, China
| | - Yuna Wang
- School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, Ningxia, China
| | - Shuya Zhang
- School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, Ningxia, China
| | - Hao Yang
- Department of Clinical Laboratory, The Second People's Hospital of Shenzhen, Shenzhen, Guangdong, China
| | - Kang Kang
- College of Medicine, Shenzhen University, Shenzhen, Guangdong, China
| | - Xinchun Chen
- College of Medicine, Shenzhen University, Shenzhen, Guangdong, China
| | - Xinying Li
- Shenzhen Geneups Biotechnology Co., Shenzhen, Guangdong, China
| | - Ming Yang
- College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, Guangdong, China
| | - Deming Gou
- College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, Guangdong, China.
| |
Collapse
|
|
18
|
Chen HC, Wang J, Shyr Y, Liu Q. FindAdapt: A python package for fast and accurate adapter detection in small RNA sequencing. PLoS Comput Biol 2024; 20:e1011786. [PMID: 38252662 PMCID: PMC10833567 DOI: 10.1371/journal.pcbi.1011786] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2023] [Revised: 02/01/2024] [Accepted: 12/26/2023] [Indexed: 01/24/2024] Open
Abstract
Adapter trimming is an essential step for analyzing small RNA sequencing data, where reads are generally longer than target RNAs ranging from 18 to 30 bp. Most adapter trimming tools require adapter information as input. However, adapter information is hard to access, specified incorrectly, or not provided with publicly available datasets, hampering their reproducibility and reusability. Manual identification of adapter patterns from raw reads is labor-intensive and error-prone. Moreover, the use of randomized adapters to reduce ligation biases during library preparation makes adapter detection even more challenging. Here, we present FindAdapt, a Python package for fast and accurate detection of adapter patterns without relying on prior information. We demonstrated that FindAdapt was far superior to existing approaches. It identified adapters successfully in 180 simulation datasets with diverse read structures and 3,184 real datasets covering a variety of commercial and customized small RNA library preparation kits. FindAdapt is stand-alone software that can be easily integrated into small RNA sequencing analysis pipelines.
Collapse
Affiliation(s)
- Hua-Chang Chen
- Department of Biostatistics, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
- Center for Quantitative Sciences, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
| | - Jing Wang
- Department of Biostatistics, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
- Center for Quantitative Sciences, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
| | - Yu Shyr
- Department of Biostatistics, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
- Center for Quantitative Sciences, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
| | - Qi Liu
- Department of Biostatistics, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
- Center for Quantitative Sciences, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
| |
Collapse
|
|
19
|
Morgunova A, Ibrahim P, Chen GG, Coury SM, Turecki G, Meaney MJ, Gifuni A, Gotlib IH, Nagy C, Ho TC, Flores C. Preparation and processing of dried blood spots for microRNA sequencing. Biol Methods Protoc 2023; 8:bpad020. [PMID: 37901452 PMCID: PMC10603595 DOI: 10.1093/biomethods/bpad020] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2023] [Revised: 09/07/2023] [Accepted: 09/19/2023] [Indexed: 10/31/2023] Open
Abstract
Dried blood spots (DBS) are biological samples commonly collected from newborns and in geographic areas distanced from laboratory settings for the purposes of disease testing and identification. MicroRNAs (miRNAs)-small non-coding RNAs that regulate gene activity at the post-transcriptional level-are emerging as critical markers and mediators of disease, including cancer, infectious diseases, and mental disorders. This protocol describes optimized procedural steps for utilizing DBS as a reliable source of biological material for obtaining peripheral miRNA expression profiles. We outline key practices, such as the method of DBS rehydration that maximizes RNA extraction yield, and the use of degenerate oligonucleotide adapters to mitigate ligase-dependent biases that are associated with small RNA sequencing. The standardization of miRNA readout from DBS offers numerous benefits: cost-effectiveness in sample collection and processing, enhanced reliability and consistency of miRNA profiling, and minimal invasiveness that facilitates repeated testing and retention of participants. The use of DBS-based miRNA sequencing is a promising method to investigate disease mechanisms and to advance personalized medicine.
Collapse
Affiliation(s)
- Alice Morgunova
- Integrated Program in Neuroscience, McGill University, Montreal, Quebec H3A 1A1, Canada
- Douglas Mental Health University Institute, McGill University, Montreal, Quebec H3A 0G4, Canada
| | - Pascal Ibrahim
- Integrated Program in Neuroscience, McGill University, Montreal, Quebec H3A 1A1, Canada
- Douglas Mental Health University Institute, McGill University, Montreal, Quebec H3A 0G4, Canada
- McGill Group for Suicide Studies, Douglas Mental Health University Institute, Verdun, Quebec H4H 1R3, Canada
| | - Gary Gang Chen
- Douglas Mental Health University Institute, McGill University, Montreal, Quebec H3A 0G4, Canada
- McGill Group for Suicide Studies, Douglas Mental Health University Institute, Verdun, Quebec H4H 1R3, Canada
| | - Saché M Coury
- Department of Psychology, Stanford University, Stanford, CA 94305, United States
- Department of Psychology, University of California, Los Angeles, CA 90095, United States
| | - Gustavo Turecki
- Douglas Mental Health University Institute, McGill University, Montreal, Quebec H3A 0G4, Canada
- McGill Group for Suicide Studies, Douglas Mental Health University Institute, Verdun, Quebec H4H 1R3, Canada
- Department of Psychiatry, McGill University, Montreal, Quebec H3A 0G4, Canada
| | - Michael J Meaney
- Douglas Mental Health University Institute, McGill University, Montreal, Quebec H3A 0G4, Canada
- Department of Psychiatry, McGill University, Montreal, Quebec H3A 0G4, Canada
- Department of Neurology and Neurosurgery, Faculty of Medicine, McGill University, Montreal, Quebec H3A 1A1, Canada
- Ludmer Centre for Neuroinformatics and Mental Health, McGill University, Montreal, Quebec H3A 2B4, Canada
- Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research (A*STAR), Brenner Centre for Molecular Medicine, Singapore City 138632, Singapore
| | - Anthony Gifuni
- Douglas Mental Health University Institute, McGill University, Montreal, Quebec H3A 0G4, Canada
- Department of Psychiatry, McGill University, Montreal, Quebec H3A 0G4, Canada
| | - Ian H Gotlib
- Department of Psychology, Stanford University, Stanford, CA 94305, United States
| | - Corina Nagy
- Douglas Mental Health University Institute, McGill University, Montreal, Quebec H3A 0G4, Canada
- McGill Group for Suicide Studies, Douglas Mental Health University Institute, Verdun, Quebec H4H 1R3, Canada
- Department of Psychiatry, McGill University, Montreal, Quebec H3A 0G4, Canada
| | - Tiffany C Ho
- Department of Psychology, Stanford University, Stanford, CA 94305, United States
- Department of Psychology, University of California, Los Angeles, CA 90095, United States
| | - Cecilia Flores
- Douglas Mental Health University Institute, McGill University, Montreal, Quebec H3A 0G4, Canada
- Department of Psychiatry, McGill University, Montreal, Quebec H3A 0G4, Canada
- Department of Neurology and Neurosurgery, Faculty of Medicine, McGill University, Montreal, Quebec H3A 1A1, Canada
- Ludmer Centre for Neuroinformatics and Mental Health, McGill University, Montreal, Quebec H3A 2B4, Canada
| |
Collapse
|
|
20
|
Mai HT, Vanness BC, Linz TH. Reverse transcription-free digital-quantitative-PCR for microRNA analysis. Analyst 2023; 148:3019-3027. [PMID: 37264955 PMCID: PMC10318481 DOI: 10.1039/d3an00351e] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/03/2023]
Abstract
MicroRNAs (miRNAs) are non-coding RNA sequences that regulate many biological processes and have become central targets of biomedical research. However, their naturally low abundances in biological samples necessitates the development of sensitive analytical techniques to conduct routine miRNA measurements in research laboratories. Digital PCR has the potential to meet this need because of its single-molecule detection capabilities, but PCR analyses of miRNAs are slowed by the ligation and reverse transcription steps first required to prepare samples. This report describes the development of a method to rapidly quantify miRNA in digital microwell arrays using base-stacking digital-quantitative-PCR (BS-dqPCR). BS-dqPCR expedites miRNA measurements by eliminating the need for ligation and reverse transcription steps, which reduces the time and cost compared to conventional miRNA PCR analyses. Under standard PCR thermocycling conditions, digital signals from miRNA samples were lower than expected, while signals from blanks were high. Therefore, a novel asymmetric thermocycling program was developed that maximized on-target signal from miRNA while minimizing non-specific amplification. The analytical response of BS-dqPCR was then evaluated over a range of miRNA concentrations. The digital PCR dimension increased in signal with increasing miRNA copy numbers. When the digital signal saturated, the quantitative PCR dimension readily discerned miRNA copy number differences. Overall, BS-dqPCR provides rapid, high-sensitivity measurements of miRNA over a wide dynamic range, which demonstrates its utility for routine miRNA analyses.
Collapse
Affiliation(s)
- Hao T Mai
- Department of Chemistry, Wayne State University, 5101 Cass Ave, Detroit, MI 48202, USA.
| | - Brice C Vanness
- Department of Chemistry, Wayne State University, 5101 Cass Ave, Detroit, MI 48202, USA.
| | - Thomas H Linz
- Department of Chemistry, Wayne State University, 5101 Cass Ave, Detroit, MI 48202, USA.
| |
Collapse
|
|
21
|
McKellar DW, Mantri M, Hinchman MM, Parker JSL, Sethupathy P, Cosgrove BD, De Vlaminck I. Spatial mapping of the total transcriptome by in situ polyadenylation. Nat Biotechnol 2023; 41:513-520. [PMID: 36329320 PMCID: PMC10110464 DOI: 10.1038/s41587-022-01517-6] [Citation(s) in RCA: 48] [Impact Index Per Article: 24.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2022] [Accepted: 09/19/2022] [Indexed: 11/05/2022]
Abstract
Spatial transcriptomics reveals the spatial context of gene expression, but current methods are limited to assaying polyadenylated (A-tailed) RNA transcripts. Here we demonstrate that enzymatic in situ polyadenylation of RNA enables detection of the full spectrum of RNAs, expanding the scope of sequencing-based spatial transcriptomics to the total transcriptome. We demonstrate that our spatial total RNA-sequencing (STRS) approach captures coding RNAs, noncoding RNAs and viral RNAs. We apply STRS to study skeletal muscle regeneration and viral-induced myocarditis. Our analyses reveal the spatial patterns of noncoding RNA expression with near-cellular resolution, identify spatially defined expression of noncoding transcripts in skeletal muscle regeneration and highlight host transcriptional responses associated with local viral RNA abundance. STRS requires adding only one step to the widely used Visium spatial total RNA-sequencing protocol from 10x Genomics, and thus could be easily adopted to enable new insights into spatial gene regulation and biology.
Collapse
Affiliation(s)
- David W McKellar
- Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
| | - Madhav Mantri
- Department of Computational Biology, Cornell University, Ithaca, NY, USA
| | - Meleana M Hinchman
- Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA
| | - John S L Parker
- Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA
| | - Praveen Sethupathy
- Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA
| | - Benjamin D Cosgrove
- Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA.
| | - Iwijn De Vlaminck
- Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA.
| |
Collapse
|
|
22
|
Green D, Singh A, Tippett VL, Tattersall L, Shah KM, Siachisumo C, Ward NJ, Thomas P, Carter S, Jeys L, Sumathi V, McNamara I, Elliott DJ, Gartland A, Dalmay T, Fraser WD. YBX1-interacting small RNAs and RUNX2 can be blocked in primary bone cancer using CADD522. J Bone Oncol 2023; 39:100474. [PMID: 36936386 PMCID: PMC10015236 DOI: 10.1016/j.jbo.2023.100474] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2023] [Revised: 02/28/2023] [Accepted: 02/28/2023] [Indexed: 03/07/2023] Open
Abstract
Primary bone cancer (PBC) comprises several subtypes each underpinned by distinctive genetic drivers. This driver diversity produces novel morphological features and clinical behaviour that serendipitously makes PBC an excellent metastasis model. Here, we report that some transfer RNA-derived small RNAs termed tRNA fragments (tRFs) perform as a constitutive tumour suppressor mechanism by blunting a potential pro-metastatic protein-RNA interaction. This mechanism is reduced in PBC progression with a gradual loss of tRNAGlyTCC cleavage into 5' end tRF-GlyTCC when comparing low-grade, intermediate-grade and high-grade patient tumours. We detected recurrent activation of miR-140 leading to upregulated RUNX2 expression in high-grade patient tumours. Both tRF-GlyTCC and RUNX2 share a sequence motif in their 3' ends that matches the YBX1 recognition site known to stabilise pro-metastatic mRNAs. Investigating some aspects of this interaction network, gain- and loss-of-function experiments using small RNA mimics and antisense LNAs, respectively, showed that ectopic tRF-GlyTCC reduced RUNX2 expression and dispersed 3D micromass architecture in vitro. iCLIP sequencing revealed YBX1 physical binding to the 3' UTR of RUNX2. The interaction between YBX1, tRF-GlyTCC and RUNX2 led to the development of the RUNX2 inhibitor CADD522 as a PBC treatment. CADD522 assessment in vitro revealed significant effects on PBC cell behaviour. In xenograft mouse models, CADD522 as a single agent without surgery significantly reduced tumour volume, increased overall and metastasis-free survival and reduced cancer-induced bone disease. Our results provide insight into PBC molecular abnormalities that have led to the identification of new targets and a new therapeutic.
Collapse
Key Words
- CADD522
- CADD522, computer aided drug design molecule 522
- CI, confidence interval
- CNV, copy number variant
- CS, chondrosarcoma
- CTC, circulating tumour cell
- DE, differentially expressed
- ES, Ewing sarcoma
- HD, high definition
- HR, hazard ratio
- OS, osteosarcoma
- RBP, RNA binding protein
- RNU6-1, U6 small nuclear 1
- ROI, region-of-interest
- Rnl, T4 RNA ligase
- SNV, single nucleotide variant
- SV, structural variant
- bone cancer
- iCLIP, individual nucleotide resolution cross-linking and immunoprecipitation
- mRNA, messenger RNA
- miRNA
- miRNA, microRNA
- piRNA, piwi interacting RNA
- sRNA, small RNA
- small RNA
- tRF
- tRF, transfer RNA fragment
- tRNA, transfer RNA
- ysRNA, Y RNA-derived sRNA
Collapse
Affiliation(s)
- Darrell Green
- Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich, UK
- Corresponding author.
| | - Archana Singh
- School of Biological Sciences, University of East Anglia, Norwich, UK
| | - Victoria L. Tippett
- The Mellanby Centre for Musculoskeletal Research, Department of Oncology and Metabolism, The University of Sheffield, UK
| | - Luke Tattersall
- The Mellanby Centre for Musculoskeletal Research, Department of Oncology and Metabolism, The University of Sheffield, UK
| | - Karan M. Shah
- The Mellanby Centre for Musculoskeletal Research, Department of Oncology and Metabolism, The University of Sheffield, UK
| | | | - Nicole J. Ward
- School of Biological Sciences, University of East Anglia, Norwich, UK
| | - Paul Thomas
- School of Biological Sciences, University of East Anglia, Norwich, UK
- Henry Wellcome Laboratory for Cell Imaging, Faculty of Science, University of East Anglia, Norwich, UK
| | - Simon Carter
- Orthopaedic Oncology, Royal Orthopaedic Hospital, Birmingham, UK
| | - Lee Jeys
- Orthopaedic Oncology, Royal Orthopaedic Hospital, Birmingham, UK
| | - Vaiyapuri Sumathi
- Musculoskeletal Pathology, University Hospitals Birmingham, Royal Orthopaedic Hospital, Birmingham, UK
| | - Iain McNamara
- Orthopaedics & Trauma, Norfolk and Norwich University Hospital, Norwich, UK
| | | | - Alison Gartland
- The Mellanby Centre for Musculoskeletal Research, Department of Oncology and Metabolism, The University of Sheffield, UK
| | - Tamas Dalmay
- School of Biological Sciences, University of East Anglia, Norwich, UK
| | - William D. Fraser
- Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich, UK
- Clinical Biochemistry, Diabetes and Endocrinology, Norfolk and Norwich University Hospital, Norwich, UK
| |
Collapse
|
|
23
|
Mok A, Tunney R, Benegas G, Wallace EWJ, Lareau LF. choros: correction of sequence-based biases for accurate quantification of ribosome profiling data. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.02.21.529452. [PMID: 36865295 PMCID: PMC9980091 DOI: 10.1101/2023.02.21.529452] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/24/2023]
Abstract
Ribosome profiling quantifies translation genome-wide by sequencing ribosome-protected fragments, or footprints. Its single-codon resolution allows identification of translation regulation, such as ribosome stalls or pauses, on individual genes. However, enzyme preferences during library preparation lead to pervasive sequence artifacts that obscure translation dynamics. Widespread over- and under-representation of ribosome footprints can dominate local footprint densities and skew estimates of elongation rates by up to five fold. To address these biases and uncover true patterns of translation, we present choros, a computational method that models ribosome footprint distributions to provide bias-corrected footprint counts. choros uses negative binomial regression to accurately estimate two sets of parameters: (i) biological contributions from codon-specific translation elongation rates; and (ii) technical contributions from nuclease digestion and ligation efficiencies. We use these parameter estimates to generate bias correction factors that eliminate sequence artifacts. Applying choros to multiple ribosome profiling datasets, we are able to accurately quantify and attenuate ligation biases to provide more faithful measurements of ribosome distribution. We show that a pattern interpreted as pervasive ribosome pausing near the beginning of coding regions is likely to arise from technical biases. Incorporating choros into standard analysis pipelines will improve biological discovery from measurements of translation.
Collapse
Affiliation(s)
- Amanda Mok
- Center for Computational Biology, University of California, Berkeley
| | - Robert Tunney
- Center for Computational Biology, University of California, Berkeley
| | - Gonzalo Benegas
- Center for Computational Biology, University of California, Berkeley
| | | | - Liana F. Lareau
- Center for Computational Biology, University of California, Berkeley
- Department of Bioengineering, University of California, Berkeley
| |
Collapse
|
|
24
|
Payet R, Billmeier M. Small RNA Profiling by Next-Generation Sequencing Using High-Definition Adapters. Methods Mol Biol 2023; 2630:103-115. [PMID: 36689179 DOI: 10.1007/978-1-0716-2982-6_8] [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] [Indexed: 01/24/2023]
Abstract
Next-generation sequencing (NGS) of small RNA (sRNA) cDNA libraries permits the identification and characterization of sRNA species de novo. However, the method through which these libraries are constructed can often introduce artifacts such as over- or underrepresentation of specific sequences or adapter oligonucleotides due to sequence biases held by the enzymes used. In this chapter we describe a protocol for sRNA library construction making use of high-definition (HD) adapters for the Illumina sequencing platform, which reduce ligation bias. This protocol leads to drastically reduced direct 5'/3' adapter ligation products and can be used for the synthesis of sRNA libraries from total RNA or sRNA of various plant, animal, and fungal samples. This protocol also includes a method for total RNA extraction from plant leaf and cultured cells or body fluids.
Collapse
Affiliation(s)
- Rocky Payet
- School of Biological Sciences, University of East Anglia, Norwich, UK.
| | - Martina Billmeier
- Institute of Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany
| |
Collapse
|
|
25
|
Chu X, He C, Sang B, Yang C, Yin C, Ji M, Qian A, Tian Y. Transfer RNAs-derived small RNAs and their application potential in multiple diseases. Front Cell Dev Biol 2022; 10:954431. [PMID: 36072340 PMCID: PMC9441921 DOI: 10.3389/fcell.2022.954431] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2022] [Accepted: 07/11/2022] [Indexed: 11/17/2022] Open
Abstract
The role of tRNAs is best known as adapter components of translational machinery. According to the central dogma of molecular biology, DNA is transcribed to RNA and in turn is translated into proteins, in which tRNA outstands by its role of the cellular courier. Recent studies have led to the revision of the canonical function of transfer RNAs (tRNAs), which indicates that tRNAs also serve as a source for short non-coding RNAs called tRNA-derived small RNAs (tsRNAs). tsRNAs play key roles in cellular processes by modulating complicated regulatory networks beyond translation and are widely involved in multiple diseases. Herein, the biogenesis and classification of tsRNAs were firstly clarified. tsRNAs are generated from pre-tRNAs or mature tRNAs and are classified into tRNA-derived fragments (tRFs) and tRNA halves (tiRNA). The tRFs include five types according to the incision loci: tRF-1, tRF-2, tRF-3, tRF-5 and i-tRF which contain 3′ tiRNA and 5′ tiRNA. The functions of tsRNAs and their regulation mechanisms involved in disease processes are systematically summarized as well. The mechanisms can elaborate on the specific regulation of tsRNAs. In conclusion, the current research suggests that tsRNAs are promising targets for modulating pathological processes, such as breast cancer, ischemic stroke, respiratory syncytial virus, osteoporosis and so on, and maintain vital clinical implications in diagnosis and therapeutics of various diseases.
Collapse
Affiliation(s)
- Xiaohua Chu
- Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering, Key Lab for Space Biosciences and Biotechnology, Research Center for Special Medicine and Health Systems Engineering, NPU-UAB Joint Laboratory for Bone Metabolism, School of Life Sciences, Northwestern Polytechnical University, Xi’an, SN, China
| | - Chenyang He
- Department of Oncology, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China
| | - Bo Sang
- Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering, Key Lab for Space Biosciences and Biotechnology, Research Center for Special Medicine and Health Systems Engineering, NPU-UAB Joint Laboratory for Bone Metabolism, School of Life Sciences, Northwestern Polytechnical University, Xi’an, SN, China
| | - Chaofei Yang
- Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering, Key Lab for Space Biosciences and Biotechnology, Research Center for Special Medicine and Health Systems Engineering, NPU-UAB Joint Laboratory for Bone Metabolism, School of Life Sciences, Northwestern Polytechnical University, Xi’an, SN, China
| | - Chong Yin
- Department of Clinical Laboratory, Academician (expert) Workstation, Lab of Epigenetics and RNA Therapy, Affiliated Hospital of North Sichuan Medical College, Nanchong, China
| | - Mili Ji
- Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering, Key Lab for Space Biosciences and Biotechnology, Research Center for Special Medicine and Health Systems Engineering, NPU-UAB Joint Laboratory for Bone Metabolism, School of Life Sciences, Northwestern Polytechnical University, Xi’an, SN, China
| | - Airong Qian
- Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering, Key Lab for Space Biosciences and Biotechnology, Research Center for Special Medicine and Health Systems Engineering, NPU-UAB Joint Laboratory for Bone Metabolism, School of Life Sciences, Northwestern Polytechnical University, Xi’an, SN, China
- *Correspondence: Airong Qian, ; Ye Tian,
| | - Ye Tian
- Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering, Key Lab for Space Biosciences and Biotechnology, Research Center for Special Medicine and Health Systems Engineering, NPU-UAB Joint Laboratory for Bone Metabolism, School of Life Sciences, Northwestern Polytechnical University, Xi’an, SN, China
- *Correspondence: Airong Qian, ; Ye Tian,
| |
Collapse
|
|
26
|
Kong X, Wang H, Guo G, Li P, Tong P, Liu M, Ma X, Dong C, Li Y, Zhang H, Zhang W. Duck sewage source coliphage P762 can lyse STEC and APEC. Virus Genes 2022; 58:436-447. [PMID: 35705841 DOI: 10.1007/s11262-022-01915-7] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2021] [Accepted: 05/10/2022] [Indexed: 11/27/2022]
Abstract
Multiple pathogenic types or serotypes restrict treatment for colibacillosis. In addition, rising antibiotic resistance has heightened public awareness to prevent and control pathogenic Escherichia coli. The bacteriophage is a viable technique to treat colibacillosis as an alternative to antibiotics. P762, a coliphage isolated from duck farm sewage, was demonstrated to cloud lyse Shiga toxin-producing Escherichia Coli serotypes O157 and non-O157 (17/39), Avian pathogenic E. coli covered serotype O78, O83, and O9 (5/19), and other pathogenic Escherichia coli (5/17). Additional fundamental biological characteristics analysis revealed that P762 is stable at pH 3 ~ 11 and temperature between 4 °C and 60 °C, and its optimum multiplicity of infection (MOI) is 0.1. The one-step curve of P762 exhibited three bursts of growth stage: two rapid and one slow stage. Furthermore, the first rapid burst size is 80 CFU/PFU, the burst size of the slow stage is 10 CFU/PFU, and the second rapid burst size is about 990 CFU/PFU. In addition, P762 can form a "halo" on a double agar plate, implying that the phage secretes depolymerase. With 95.14% identity and 90% query coverage, genome sequence analysis revealed that P762 is most closely related to Escherichia phage DY1, which belongs to the genus Kayfunavirus. After screening using RAST and VFDB, no virulence factors were discovered in P762. In vitro antibacterial tests revealed that P762 has high bactericidal activity in lettuce leaves contaminated with STEC. In conclusion, phage P762 might be employed in the future to prevent and control pathogenic Escherichia coli.
Collapse
Affiliation(s)
- Xuewei Kong
- College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China
- Key Lab of Animal Bacteriology, Ministry of Agriculture, Nanjing, China
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China
| | - Hui Wang
- College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China
- Key Lab of Animal Bacteriology, Ministry of Agriculture, Nanjing, China
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China
| | - Genglin Guo
- College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China
- Key Lab of Animal Bacteriology, Ministry of Agriculture, Nanjing, China
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China
| | - Pei Li
- College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China
- Key Lab of Animal Bacteriology, Ministry of Agriculture, Nanjing, China
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China
| | - Panpan Tong
- College of Veterinary Medicine, Shihezi Agricultural University, Xinjiang, China
| | - Maojun Liu
- Jiangsu Academy of Agricultural Sciences, Nanjing, China
| | - Xun Ma
- College of Animal Science and Technology, Xinjiang Agricultural University, Xinjiang, China
| | - Chen Dong
- Jiangsu Province CDC: Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, China
| | - Yubao Li
- Agricultural Science and Engineering School, Liaocheng University, Liaocheng, China
| | - Haiyan Zhang
- Department of Food and Biology Engineering, Wuhu Institute of Technology, Wuhu, China.
| | - Wei Zhang
- College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
- Key Lab of Animal Bacteriology, Ministry of Agriculture, Nanjing, China.
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
| |
Collapse
|
|
27
|
Zhou Y, Sotcheff SL, Routh AL. Next-generation sequencing: A new avenue to understand viral RNA-protein interactions. J Biol Chem 2022; 298:101924. [PMID: 35413291 PMCID: PMC8994257 DOI: 10.1016/j.jbc.2022.101924] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2021] [Revised: 04/01/2022] [Accepted: 04/02/2022] [Indexed: 10/25/2022] Open
Abstract
The genomes of RNA viruses present an astonishing source of both sequence and structural diversity. From intracellular viral RNA-host interfaces to interactions between the RNA genome and structural proteins in virus particles themselves, almost the entire viral lifecycle is accompanied by a myriad of RNA-protein interactions that are required to fulfill their replicative potential. It is therefore important to characterize such rich and dynamic collections of viral RNA-protein interactions to understand virus evolution and their adaptation to their hosts and environment. Recent advances in next-generation sequencing technologies have allowed the characterization of viral RNA-protein interactions, including both transient and conserved interactions, where molecular and structural approaches have fallen short. In this review, we will provide a methodological overview of the high-throughput techniques used to study viral RNA-protein interactions, their biochemical mechanisms, and how they evolved from classical methods as well as one another. We will discuss how different techniques have fueled virus research to characterize how viral RNA and proteins interact, both locally and on a global scale. Finally, we will present examples on how these techniques influence the studies of clinically important pathogens such as HIV-1 and SARS-CoV-2.
Collapse
Affiliation(s)
- Yiyang Zhou
- Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, Texas, USA.
| | - Stephanea L Sotcheff
- Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, Texas, USA
| | - Andrew L Routh
- Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, Texas, USA; Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, Texas, USA; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, Texas, USA
| |
Collapse
|
|
28
|
Chen H, Alonso JM, Stepanova AN. A Ribo-Seq Method to Study Genome-Wide Translational Regulation in Plants. METHODS IN MOLECULAR BIOLOGY (CLIFTON, N.J.) 2022; 2494:61-98. [PMID: 35467201 DOI: 10.1007/978-1-0716-2297-1_6] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Protein production from mRNA is one of the fundamental molecular processes in a cell. Accurate genome-wide information on the levels of translation and ribosome distribution on mRNA can be gathered by carrying out ribosome footprinting, aka Ribo-seq. Herein, we present a detailed protocol describing the construction of parallel Ribo-seq and RNA-seq libraries from Arabidopsis seedlings treated with the plant hormone auxin. The improved protocol for ribosome footprint library generation can be easily adapted to analyzing the effects on translation of genetic perturbations and various abiotic and biotic factors to shed the much-needed light on translational regulation in plants.
Collapse
Affiliation(s)
- Hao Chen
- Department of Plant and Microbial Biology, Program in Genetics, North Carolina State University, Raleigh, NC, USA
| | - Jose M Alonso
- Department of Plant and Microbial Biology, Program in Genetics, North Carolina State University, Raleigh, NC, USA
| | - Anna N Stepanova
- Department of Plant and Microbial Biology, Program in Genetics, North Carolina State University, Raleigh, NC, USA.
| |
Collapse
|
|
29
|
Bornelöv S, Czech B, Hannon GJ. An evolutionarily conserved stop codon enrichment at the 5' ends of mammalian piRNAs. Nat Commun 2022; 13:2118. [PMID: 35440552 PMCID: PMC9018710 DOI: 10.1038/s41467-022-29787-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2021] [Accepted: 03/03/2022] [Indexed: 11/10/2022] Open
Abstract
PIWI-interacting RNAs (piRNAs) are small RNAs required to recognize and silence transposable elements. The 5' ends of mature piRNAs are defined through cleavage of long precursor transcripts, primarily by Zucchini (Zuc). Zuc-dependent cleavage typically occurs immediately upstream of a uridine. However, Zuc lacks sequence preference in vitro, pointing towards additional unknown specificity factors. Here, we examine murine piRNAs and reveal a strong and specific enrichment of three sequences (UAA, UAG, UGA)-corresponding to stop codons-at piRNA 5' ends. Stop codon sequences are also enriched immediately after piRNA processing intermediates, reflecting their Zuc-dependent tail-to-head arrangement. Further analyses reveal that a Zuc in vivo cleavage preference at four sequences (UAA, UAG, UGA, UAC) promotes 5' end stop codons. This observation is conserved across mammals and possibly further. Our work provides new insights into Zuc-dependent cleavage and may point to a previously unrecognized connection between piRNA biogenesis and the translational machinery.
Collapse
Affiliation(s)
- Susanne Bornelöv
- Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, CB2 0RE, UK.
| | - Benjamin Czech
- Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, CB2 0RE, UK
| | - Gregory J Hannon
- Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, CB2 0RE, UK.
| |
Collapse
|
|
30
|
Shi J, Zhou T, Chen Q. Exploring the expanding universe of small RNAs. Nat Cell Biol 2022; 24:415-423. [PMID: 35414016 PMCID: PMC9035129 DOI: 10.1038/s41556-022-00880-5] [Citation(s) in RCA: 110] [Impact Index Per Article: 36.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2021] [Accepted: 03/02/2022] [Indexed: 12/11/2022]
Abstract
The world of small noncoding RNAs (sncRNAs) is ever-expanding, from small interfering RNA, microRNA and Piwi-interacting RNA to the recently emerging non-canonical sncRNAs derived from longer structured RNAs (for example, transfer, ribosomal, Y, small nucleolar, small nuclear and vault RNAs), showing distinct biogenesis and functional principles. Here we discuss recent tools for sncRNA identification, caveats in sncRNA expression analysis and emerging methods for direct sequencing of sncRNAs and systematic mapping of RNA modifications that are integral to their function.
Collapse
Affiliation(s)
- Junchao Shi
- Division of Biomedical Sciences, School of Medicine, University of California, Riverside, CA, USA
| | - Tong Zhou
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA.
| | - Qi Chen
- Division of Biomedical Sciences, School of Medicine, University of California, Riverside, CA, USA.
| |
Collapse
|
|
31
|
Small RNA-Sequencing for Analysis of Circulating miRNAs: Benchmark Study. J Mol Diagn 2022; 24:386-394. [PMID: 35081459 DOI: 10.1016/j.jmoldx.2021.12.006] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2021] [Revised: 11/15/2021] [Accepted: 12/20/2021] [Indexed: 11/22/2022] Open
Abstract
Small RNA-sequencing (RNA-Seq) is being increasingly used for profiling of circulating microRNAs (miRNAs), a new group of promising biomarkers. Unfortunately, small RNA-Seq protocols are prone to biases limiting quantification accuracy, which motivated development of several novel methods. Here, we present comparison of all small RNA-Seq library preparation approaches that are commercially available for quantification of miRNAs in biofluids. Using synthetic and human plasma samples, we compared performance of traditional two-adaptor ligation protocols (Lexogen, Norgen), as well as methods using randomized adaptors (NEXTflex), polyadenylation (SMARTer), circularization (RealSeq), capture probes (EdgeSeq), or unique molecular identifiers (QIAseq). There was no single protocol outperforming others across all metrics. Limited overlap of measured miRNA profiles was documented between methods largely owing to protocol-specific biases. Methods designed to minimize bias largely differ in their performance, and contributing factors were identified. Usage of unique molecular identifiers has rather negligible effect and, if designed incorrectly, can even introduce spurious results. Together, these results identify strengths and weaknesses of all current methods and provide guidelines for applications of small RNA-Seq in biomarker research.
Collapse
|
|
32
|
A comparative analysis of single cell small RNA sequencing data reveals heterogeneous isomiR expression and regulation. Sci Rep 2022; 12:2834. [PMID: 35181712 PMCID: PMC8857176 DOI: 10.1038/s41598-022-06876-3] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2021] [Accepted: 01/31/2022] [Indexed: 12/12/2022] Open
Abstract
MicroRNAs (miRNAs) are non-coding small RNAs which play a critical role in the regulation of gene expression in cells. It is known that miRNAs are often expressed as multiple isoforms, called isomiRs, which may have alternative regulatory functions. Despite the recent development of several single cell small RNA sequencing protocols, these methods have not been leveraged to investigate isomiR expression and regulation to better understand their role on a single cell level. Here we integrate sequencing data from three independent studies and find substantial differences in isomiR composition that suggest that cell autonomous mechanisms may drive isomiR processing. We also find evidence of altered regulatory functions of different classes of isomiRs, when compared to their respective wild-type miRNA, which supports a biological role for many of the isomiRs that are expressed.
Collapse
|
|
33
|
Maguire S, Guan S. Rolling circle reverse transcription enables high fidelity nanopore sequencing of small RNA. PLoS One 2022; 17:e0275471. [PMID: 36215256 PMCID: PMC9550094 DOI: 10.1371/journal.pone.0275471] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2022] [Accepted: 09/17/2022] [Indexed: 11/07/2022] Open
Abstract
Small RNAs (sRNAs) are an important group of non-coding RNAs that have great potential as diagnostic and prognostic biomarkers for treatment of a wide variety of diseases. The portability and affordability of nanopore sequencing technology makes it ideal for point of care and low resource settings. Currently sRNAs can't be reliably sequenced on the nanopore platform due to the short size of sRNAs and high error rate of the nanopore sequencer. Here, we developed a highly efficient nanopore-based sequencing strategy for sRNAs (SR-Cat-Seq) in which sRNAs are ligated to an adapter, circularized, and undergo rolling circle reverse transcription to generate concatemeric cDNA. After sequencing, the resulting tandem repeat sequences within the individual cDNA can be aligned to generate highly accurate consensus sequences. We compared our sequencing strategy with other sRNA sequencing methods on a short-read sequencing platform and demonstrated that SR-Cat-Seq can obtain low bias and highly accurate sRNA transcriptomes. Therefore, our method could enable nanopore sequencing for sRNA-based diagnostics and other applications.
Collapse
Affiliation(s)
- Sean Maguire
- New England Biolabs, Inc., Beverly, MA, United States of America
| | - Shengxi Guan
- New England Biolabs, Inc., Beverly, MA, United States of America
- * E-mail:
| |
Collapse
|
|
34
|
Kugelberg U, Nätt D, Skog S, Kutter C, Öst A. 5´XP sRNA-seq: efficient identification of transcripts with and without 5´ phosphorylation reveals evolutionary conserved small RNA. RNA Biol 2021; 18:1588-1599. [PMID: 33382953 PMCID: PMC8594926 DOI: 10.1080/15476286.2020.1861770] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2020] [Revised: 12/04/2020] [Accepted: 12/06/2020] [Indexed: 12/21/2022] Open
Abstract
Small RNA (sRNA) sequencing has been critical for our understanding of many cellular processes, including gene regulation. Nonetheless, the varying biochemical properties of sRNA, such as 5´ nucleotide modifications, make many sRNA subspecies incompatible with common protocols for sRNA sequencing. Here we describe 5XP-seq that outlines a novel strategy that captures a more complete picture of sRNA. By tagging 5´P sRNA during library preparation, 5XP-seq combines an open approach that includes all types of 5'-terminal modifications (5´X), with a selective approach for 5-phosphorylated sRNA (5´P). We show that 5XP-seq not only enriches phosphorylated miRNA and piRNA but successfully discriminates these sRNA from all other sRNA species. We further demonstrate the importance of this strategy by successful inter-species validation of sRNAs that would have otherwise failed, including human to insect translation of several tRNA (tRFs) and rRNA (rRFs) fragments. By combining 5´ insensitive library strategies with 5´ sensitive tagging, we have successfully tackled an intrinsic bias in modern sRNA sequencing that will help us reveal the true complexity and the evolutionary significance of the sRNA world.
Collapse
Affiliation(s)
- Unn Kugelberg
- Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden
| | - Daniel Nätt
- Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden
| | - Signe Skog
- Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden
| | - Claudia Kutter
- Department of Microbiology, Tumor and Cell Biology, Science for Life Laboratory, Karolinska Institute, Stockholm
| | - Anita Öst
- Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden
| |
Collapse
|
|
35
|
Wulf MG, Maguire S, Dai N, Blondel A, Posfai D, Krishnan K, Sun Z, Guan S, Corrêa IR. Chemical capping improves template switching and enhances sequencing of small RNAs. Nucleic Acids Res 2021; 50:e2. [PMID: 34581823 PMCID: PMC8754658 DOI: 10.1093/nar/gkab861] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2021] [Revised: 08/26/2021] [Accepted: 09/14/2021] [Indexed: 12/16/2022] Open
Abstract
Template-switching reverse transcription is widely used in RNA sequencing for low-input and low-quality samples, including RNA from single cells or formalin-fixed paraffin-embedded (FFPE) tissues. Previously, we identified the native eukaryotic mRNA 5′ cap as a key structural element for enhancing template switching efficiency. Here, we introduce CapTS-seq, a new strategy for sequencing small RNAs that combines chemical capping and template switching. We probed a variety of non-native synthetic cap structures and found that an unmethylated guanosine triphosphate cap led to the lowest bias and highest efficiency for template switching. Through cross-examination of different nucleotides at the cap position, our data provided unequivocal evidence that the 5′ cap acts as a template for the first nucleotide in reverse transcriptase-mediated post-templated addition to the emerging cDNA—a key feature to propel template switching. We deployed CapTS-seq for sequencing synthetic miRNAs, human total brain and liver FFPE RNA, and demonstrated that it consistently improves library quality for miRNAs in comparison with a gold standard template switching-based small RNA-seq kit.
Collapse
Affiliation(s)
- Madalee G Wulf
- New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA
| | - Sean Maguire
- New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA
| | - Nan Dai
- New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA
| | - Alice Blondel
- New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA
| | - Dora Posfai
- New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA
| | | | - Zhiyi Sun
- New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA
| | - Shengxi Guan
- New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA
| | - Ivan R Corrêa
- New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA
| |
Collapse
|
|
36
|
Abstract
Transcription start site (TSS) selection influences transcript stability and translation as well as protein sequence. Alternative TSS usage is pervasive in organismal development, is a major contributor to transcript isoform diversity in humans, and is frequently observed in human diseases including cancer. In this review, we discuss the breadth of techniques that have been used to globally profile TSSs and the resulting insights into gene regulation, as well as future prospects in this area of inquiry.
Collapse
Affiliation(s)
| | - Gabriel E. Zentner
- Department of Biology, Indiana University, Bloomington, IN 47401, USA
- Indiana University Melvin and Bren Simon Comprehensive Cancer Center, Indianapolis, IN 46202, USA
| |
Collapse
|
|
37
|
Hu JF, Yim D, Ma D, Huber SM, Davis N, Bacusmo JM, Vermeulen S, Zhou J, Begley TJ, DeMott MS, Levine SS, de Crécy-Lagard V, Dedon PC, Cao B. Quantitative mapping of the cellular small RNA landscape with AQRNA-seq. Nat Biotechnol 2021; 39:978-988. [PMID: 33859402 PMCID: PMC8355021 DOI: 10.1038/s41587-021-00874-y] [Citation(s) in RCA: 47] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2019] [Accepted: 02/25/2021] [Indexed: 12/23/2022]
Abstract
Current next-generation RNA-sequencing (RNA-seq) methods do not provide accurate quantification of small RNAs within a sample, due to sequence-dependent biases in capture, ligation and amplification during library preparation. We present a method, absolute quantification RNA-sequencing (AQRNA-seq), that minimizes biases and provides a direct, linear correlation between sequencing read count and copy number for all small RNAs in a sample. Library preparation and data processing were optimized and validated using a 963-member microRNA reference library, oligonucleotide standards of varying length, and RNA blots. Application of AQRNA-seq to a panel of human cancer cells revealed >800 detectable miRNAs that varied during cancer progression, while application to bacterial transfer RNA pools, with the challenges of secondary structure and abundant modifications, revealed 80-fold variation in tRNA isoacceptor levels, stress-induced site-specific tRNA fragmentation, quantitative modification maps, and evidence for stress-induced, tRNA-driven, codon-biased translation. AQRNA-seq thus provides a versatile means to quantitatively map the small RNA landscape in cells.
Collapse
Affiliation(s)
- Jennifer F Hu
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA
- Bristol Myers Squibb, Seattle, WA, USA
| | - Daniel Yim
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
- A*STAR Genome Institute of Singapore, Singapore, Singapore
| | - Duanduan Ma
- BioMicro Center, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Sabrina M Huber
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
- Laboratory of Toxicology, ETH Zürich, Zürich, Switzerland
| | - Nick Davis
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
- Theon Therapeutics, Cambridge, MA, USA
| | - Jo Marie Bacusmo
- Department of Microbiology & Cell Science, University of Florida, Gainesville, FL, USA
| | - Sidney Vermeulen
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Jieliang Zhou
- KK Research Center, KK Women's and ChildrenBristol Myers Squibb's Hospital, Singapore, Singapore
| | - Thomas J Begley
- The RNA Institute and Department of Biology, University at Albany, Albany, NY, USA
| | - Michael S DeMott
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
- Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Stuart S Levine
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
- BioMicro Center, Massachusetts Institute of Technology, Cambridge, MA, USA
- Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA
| | | | - Peter C Dedon
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
- Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA.
- Singapore-MIT Alliance for Research and Technology Antimicrobial Resistance IRG, Singapore, Singapore.
| | - Bo Cao
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
- Singapore-MIT Alliance for Research and Technology Antimicrobial Resistance IRG, Singapore, Singapore.
- College of Life Sciences, Qufu Normal University, Qufu, China.
| |
Collapse
|
|
38
|
Fabry MH, Falconio FA, Joud F, Lythgoe EK, Czech B, Hannon GJ. Maternally inherited piRNAs direct transient heterochromatin formation at active transposons during early Drosophila embryogenesis. eLife 2021; 10:e68573. [PMID: 34236313 PMCID: PMC8352587 DOI: 10.7554/elife.68573] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2021] [Accepted: 07/07/2021] [Indexed: 12/12/2022] Open
Abstract
The PIWI-interacting RNA (piRNA) pathway controls transposon expression in animal germ cells, thereby ensuring genome stability over generations. In Drosophila, piRNAs are intergenerationally inherited through the maternal lineage, and this has demonstrated importance in the specification of piRNA source loci and in silencing of I- and P-elements in the germ cells of daughters. Maternally inherited Piwi protein enters somatic nuclei in early embryos prior to zygotic genome activation and persists therein for roughly half of the time required to complete embryonic development. To investigate the role of the piRNA pathway in the embryonic soma, we created a conditionally unstable Piwi protein. This enabled maternally deposited Piwi to be cleared from newly laid embryos within 30 min and well ahead of the activation of zygotic transcription. Examination of RNA and protein profiles over time, and correlation with patterns of H3K9me3 deposition, suggests a role for maternally deposited Piwi in attenuating zygotic transposon expression in somatic cells of the developing embryo. In particular, robust deposition of piRNAs targeting roo, an element whose expression is mainly restricted to embryonic development, results in the deposition of transient heterochromatic marks at active roo insertions. We hypothesize that roo, an extremely successful mobile element, may have adopted a lifestyle of expression in the embryonic soma to evade silencing in germ cells.
Collapse
Affiliation(s)
- Martin H Fabry
- CRUK Cambridge Institute, University of Cambridge, Li Ka Shing CentreCambridgeUnited Kingdom
| | - Federica A Falconio
- CRUK Cambridge Institute, University of Cambridge, Li Ka Shing CentreCambridgeUnited Kingdom
| | - Fadwa Joud
- CRUK Cambridge Institute, University of Cambridge, Li Ka Shing CentreCambridgeUnited Kingdom
| | - Emily K Lythgoe
- CRUK Cambridge Institute, University of Cambridge, Li Ka Shing CentreCambridgeUnited Kingdom
| | - Benjamin Czech
- CRUK Cambridge Institute, University of Cambridge, Li Ka Shing CentreCambridgeUnited Kingdom
| | - Gregory J Hannon
- CRUK Cambridge Institute, University of Cambridge, Li Ka Shing CentreCambridgeUnited Kingdom
| |
Collapse
|
|
39
|
Rose SA, Wroblewska A, Dhainaut M, Yoshida H, Shaffer JM, Bektesevic A, Ben-Zvi B, Rhoads A, Kim EY, Yu B, Lavin Y, Merad M, Buenrostro JD, Brown BD. A microRNA expression and regulatory element activity atlas of the mouse immune system. Nat Immunol 2021; 22:914-927. [PMID: 34099919 PMCID: PMC8480231 DOI: 10.1038/s41590-021-00944-y] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2020] [Accepted: 04/26/2021] [Indexed: 02/05/2023]
Abstract
To better define the control of immune system regulation, we generated an atlas of microRNA (miRNA) expression from 63 mouse immune cell populations and connected these signatures with assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation followed by sequencing (ChIP-seq) and nascent RNA profiles to establish a map of miRNA promoter and enhancer usage in immune cells. miRNA complexity was relatively low, with >90% of the miRNA compartment of each population comprising <75 miRNAs; however, each cell type had a unique miRNA signature. Integration of miRNA expression with chromatin accessibility revealed putative regulatory elements for differentially expressed miRNAs, including miR-21a, miR-146a and miR-223. The integrated maps suggest that many miRNAs utilize multiple promoters to reach high abundance and identified dominant and divergent miRNA regulatory elements between lineages and during development that may be used by clustered miRNAs, such as miR-99a/let-7c/miR-125b, to achieve distinct expression. These studies, with web-accessible data, help delineate the cis-regulatory elements controlling miRNA signatures of the immune system.
Collapse
Affiliation(s)
- Samuel A Rose
- Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Aleksandra Wroblewska
- Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Maxime Dhainaut
- Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Hideyuki Yoshida
- YCI Laboratory for Immunological Transcriptomics, RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan
| | | | - Anela Bektesevic
- Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Benjamin Ben-Zvi
- Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Andrew Rhoads
- Department of Pathology, Harvard Medical School, Boston, MA, USA
| | - Edy Y Kim
- Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Boston, MA, USA
- Harvard Medical School, Boston, MA, USA
| | - Bingfei Yu
- Division of Biology, University of California San Diego, La Jolla, CA, USA
| | - Yonit Lavin
- Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Miriam Merad
- Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Jason D Buenrostro
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA
| | - Brian D Brown
- Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
| |
Collapse
|
|
40
|
Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs. BMC Biol 2021; 19:129. [PMID: 34158037 PMCID: PMC8220740 DOI: 10.1186/s12915-021-01053-w] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2021] [Accepted: 05/24/2021] [Indexed: 12/13/2022] Open
Abstract
Background Targeted installation of designer chemical moieties on biopolymers provides an orthogonal means for their visualisation, manipulation and sequence analysis. Although high-throughput RNA sequencing is a widely used method for transcriptome analysis, certain steps, such as 3′ adapter ligation in strand-specific RNA sequencing, remain challenging due to structure- and sequence-related biases introduced by RNA ligases, leading to misrepresentation of particular RNA species. Here, we remedy this limitation by adapting two RNA 2′-O-methyltransferases from the Hen1 family for orthogonal chemo-enzymatic click tethering of a 3′ sequencing adapter that supports cDNA production by reverse transcription of the tagged RNA. Results We showed that the ssRNA-specific DmHen1 and dsRNA-specific AtHEN1 can be used to efficiently append an oligonucleotide adapter to the 3′ end of target RNA for sequencing library preparation. Using this new chemo-enzymatic approach, we identified miRNAs and prokaryotic small non-coding sRNAs in probiotic Lactobacillus casei BL23. We found that compared to a reference conventional RNA library preparation, methyltransferase-Directed Orthogonal Tagging and RNA sequencing, mDOT-seq, avoids misdetection of unspecific highly-structured RNA species, thus providing better accuracy in identifying the groups of transcripts analysed. Our results suggest that mDOT-seq has the potential to advance analysis of eukaryotic and prokaryotic ssRNAs. Conclusions Our findings provide a valuable resource for studies of the RNA-centred regulatory networks in Lactobacilli and pave the way to developing novel transcriptome and epitranscriptome profiling approaches in vitro and inside living cells. As RNA methyltransferases share the structure of the AdoMet-binding domain and several specific cofactor binding features, the basic principles of our approach could be easily translated to other AdoMet-dependent enzymes for the development of modification-specific RNA-seq techniques. Supplementary Information The online version contains supplementary material available at 10.1186/s12915-021-01053-w.
Collapse
|
|
41
|
Benesova S, Kubista M, Valihrach L. Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis. Diagnostics (Basel) 2021; 11:964. [PMID: 34071824 PMCID: PMC8229417 DOI: 10.3390/diagnostics11060964] [Citation(s) in RCA: 47] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2021] [Revised: 05/21/2021] [Accepted: 05/24/2021] [Indexed: 01/15/2023] Open
Abstract
MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol's performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.
Collapse
Affiliation(s)
- Sarka Benesova
- Laboratory of Gene Expression, Institute of Biotechnology, CAS, BIOCEV, 252 50 Vestec, Czech Republic; (S.B.); (M.K.)
- Laboratory of Informatics and Chemistry, Faculty of Chemical Technology, University of Chemistry and Technology, 166 28 Prague, Czech Republic
| | - Mikael Kubista
- Laboratory of Gene Expression, Institute of Biotechnology, CAS, BIOCEV, 252 50 Vestec, Czech Republic; (S.B.); (M.K.)
- TATAA Biocenter AB, 411 03 Gothenburg, Sweden
| | - Lukas Valihrach
- Laboratory of Gene Expression, Institute of Biotechnology, CAS, BIOCEV, 252 50 Vestec, Czech Republic; (S.B.); (M.K.)
| |
Collapse
|
|
42
|
Wang H, Huang R, Li L, Zhu J, Li Z, Peng C, Zhuang X, Lin H, Shi S, Huang P. CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini. Cell Discov 2021; 7:25. [PMID: 33867522 PMCID: PMC8053708 DOI: 10.1038/s41421-021-00265-2] [Citation(s) in RCA: 38] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2021] [Accepted: 03/25/2021] [Indexed: 12/20/2022] Open
Abstract
High-throughput sequencing reveals the complex landscape of small noncoding RNAs (sRNAs). However, it is limited by requiring 5'-monophosphate and 3'-hydroxyl in RNAs for adapter ligation and hindered by methylated nucleosides that interfere with reverse transcription. Here we develop Cap-Clip acid pyrophosphatase (Cap-Clip), T4 polynucleotide kinase (PNK) and AlkB/AlkB(D135S)-facilitated small ncRNA sequencing (CPA-seq) to detect and quantify sRNAs with terminus multiplicities and nucleoside methylations. CPA-seq identified a large number of previously undetected sRNAs. Comparison of sRNAs with or without AlkB/AlkB(D135S) treatment reveals nucleoside methylations on sRNAs. Using CPA-seq, we profiled the sRNA transcriptomes (sRNomes) of nine mouse tissues and reported the extensive tissue-specific differences of sRNAs. We also observed the transition of sRNomes during hepatic reprogramming. Knockdown of mesenchymal stem cell-enriched U1-5' snsRNA promoted hepatic reprogramming. CPA-seq is a powerful tool with high sensitivity and specificity for profiling sRNAs with methylated nucleosides and diverse termini.
Collapse
Affiliation(s)
- Heming Wang
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China.,University of Chinese Academy of Sciences, Beijing, 100049, China.,CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China
| | - Rong Huang
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China.,University of Chinese Academy of Sciences, Beijing, 100049, China.,CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China
| | - Ling Li
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
| | - Junjin Zhu
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China.,CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China
| | - Zhihong Li
- National Facility for Protein Science in Shanghai, Zhangjiang Lab, Shanghai Advanced Research Institute, Chinese Academy of Science, Shanghai, 201210, China
| | - Chao Peng
- National Facility for Protein Science in Shanghai, Zhangjiang Lab, Shanghai Advanced Research Institute, Chinese Academy of Science, Shanghai, 201210, China
| | - Xuran Zhuang
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
| | - Haifan Lin
- Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, New Haven, CT, 06520, USA
| | - Shuo Shi
- Shanghai Institute for Advanced Immunochemical Studies (SIAIS), ShanghaiTech University, Shanghai, 201210, China.
| | - Pengyu Huang
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. .,CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China. .,Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, 300192, China.
| |
Collapse
|
|
43
|
Chen CH, Lu F, Yang WJ, Yang PE, Chen WM, Kang ST, Huang YS, Kao YC, Feng CT, Chang PC, Wang T, Hsieh CA, Lin YC, Jen Huang JY, Wang LHC. A novel platform for discovery of differentially expressed microRNAs in patients with repeated implantation failure. Fertil Steril 2021; 116:181-188. [PMID: 33823989 DOI: 10.1016/j.fertnstert.2021.01.055] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2020] [Revised: 01/29/2021] [Accepted: 01/29/2021] [Indexed: 12/22/2022]
Abstract
OBJECTIVE To identify predictor microRNAs (miRNAs) from patients with repeated implantation failure (RIF). DESIGN Systemic analysis of miRNA profiles from the endometrium of patients undergoing in vitro fertilization (IVF). SETTING University research institute, private IVF center, and molecular testing laboratory. PATIENT(S) Twenty five infertile patients in the discovery cohort and 11 patients in the validation cohort. INTERVENTIONS(S) None. MAIN OUTCOME MEASURE(S) A signature set of miRNA associated with the risk of RIF. RESULT(S) We designed a reproductive disease-related PanelChip to access endometrium miRNA profiles in patients undergoing IVF. Three major miRNA signatures, including hsa-miR-20b-5p, hsa-miR-155-5p, and hsa-miR-718, were identified using infinite combination signature search algorithm analysis from 25 patients in the discovery cohort undergoing IVF. These miRNAs were used as biomarkers in the validation cohort of 11 patients. Finally, the 3-miRNA signature was capable of predicting patients with RIF with an accuracy >90%. CONCLUSION(S) Our findings indicated that specific endometrial miRNAs can be applied as diagnostic biomarkers to predict RIF. Such information will definitely help to increase the success rate of implantation practice.
Collapse
Affiliation(s)
- Ching Hung Chen
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan; Department of Obstetrics and Gynecology, Ton Yen General Hospital, Hsinchu, Taiwan; Taiwan IVF Group Center for Reproductive Medicine and Infertility, Hsinchu, Taiwan
| | - Farn Lu
- Department of Obstetrics and Gynecology, Ton Yen General Hospital, Hsinchu, Taiwan; Taiwan IVF Group Center for Reproductive Medicine and Infertility, Hsinchu, Taiwan
| | - Wen Jui Yang
- Department of Obstetrics and Gynecology, Ton Yen General Hospital, Hsinchu, Taiwan; Taiwan IVF Group Center for Reproductive Medicine and Infertility, Hsinchu, Taiwan
| | | | | | | | | | - Yi Chi Kao
- Quark Biosciences, Inc., Hsinchu, Taiwan
| | | | | | | | - Chi An Hsieh
- Taiwan IVF Group Center for Reproductive Medicine and Infertility, Hsinchu, Taiwan
| | - Yu Chun Lin
- Taiwan IVF Group Center for Reproductive Medicine and Infertility, Hsinchu, Taiwan
| | - Jack Yu Jen Huang
- Department of Obstetrics and Gynecology, Ton Yen General Hospital, Hsinchu, Taiwan; Taiwan IVF Group Center for Reproductive Medicine and Infertility, Hsinchu, Taiwan; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Stanford University, Stanford, California
| | - Lily Hui-Ching Wang
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan; Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan.
| |
Collapse
|
|
44
|
van Dijk EL, Thermes C. A Small RNA-Seq Protocol with Less Bias and Improved Capture of 2'-O-Methyl RNAs. Methods Mol Biol 2021; 2298:153-167. [PMID: 34085244 DOI: 10.1007/978-1-0716-1374-0_10] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNAs such as plant microRNAs (miRNAs) carry a 2'-O-methyl (2'-OMe) modification at their 3' terminal nucleotide. This modification adds another level of difficulty as it inhibits 3' adapter ligation. We previously demonstrated that modified versions of the "TruSeq (TS)" protocol have less bias and an improved detection of 2'-OMe RNAs. Here we describe in detail protocol "TS5," which showed the best overall performance. We also provide guidelines for bioinformatics analysis of the sequencing data.
Collapse
Affiliation(s)
- Erwin L van Dijk
- Institute for Integrative Biology of the Cell, UMR9198, CNRS CEA Univ Paris-Sud, Université Paris-Saclay, Gif sur Yvette Cedex, France.
| | - Claude Thermes
- Institute for Integrative Biology of the Cell, UMR9198, CNRS CEA Univ Paris-Sud, Université Paris-Saclay, Gif sur Yvette Cedex, France
| |
Collapse
|
|
45
|
Xie W, Sowemimo I, Hayashi R, Wang J, Burkard TR, Brennecke J, Ameres SL, Patel DJ. Structure-function analysis of microRNA 3'-end trimming by Nibbler. Proc Natl Acad Sci U S A 2020; 117:30370-30379. [PMID: 33199607 PMCID: PMC7720153 DOI: 10.1073/pnas.2018156117] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Nibbler (Nbr) is a 3'-to-5' exoribonuclease whose catalytic 3'-end trimming activity impacts microRNA (miRNA) and PIWI-interacting RNA (piRNA) biogenesis. Here, we report on structural and functional studies to decipher the contributions of Nbr's N-terminal domain (NTD) and exonucleolytic domain (EXO) in miRNA 3'-end trimming. We have solved the crystal structures of the NTD core and EXO domains of Nbr, both in the apo-state. The NTD-core domain of Aedes aegypti Nbr adopts a HEAT-like repeat scaffold with basic patches constituting an RNA-binding surface exhibiting a preference for binding double-strand RNA (dsRNA) over single-strand RNA (ssRNA). Structure-guided functional assays in Drosophila S2 cells confirmed a principal role of the NTD in exonucleolytic miRNA trimming, which depends on basic surface patches. Gain-of-function experiments revealed a potential role of the NTD in recruiting Nbr to Argonaute-bound small RNA substrates. The EXO domain of A. aegypti and Drosophila melanogaster Nbr adopt a mixed α/β-scaffold with a deep pocket lined by a DEDDy catalytic cleavage motif. We demonstrate that Nbr's EXO domain exhibits Mn2+-dependent ssRNA-specific 3'-to-5' exoribonuclease activity. Modeling of a 3' terminal Uridine into the catalytic pocket of Nbr EXO indicates that 2'-O-methylation of the 3'-U would result in a steric clash with a tryptophan side chain, suggesting that 2'-O-methylation protects small RNAs from Nbr-mediated trimming. Overall, our data establish that Nbr requires its NTD as a substrate recruitment platform to execute exonucleolytic miRNA maturation, catalyzed by the ribonuclease EXO domain.
Collapse
Affiliation(s)
- Wei Xie
- Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065
| | - Ivica Sowemimo
- Institute of Molecular Biotechnology, Vienna BioCenter, 1030 Vienna, Austria
| | - Rippei Hayashi
- Department of Genome Sciences, The John Curtin School of Medical Research, Australian National University, Canberra 2601, Australia
| | - Juncheng Wang
- Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065
| | - Thomas R Burkard
- Institute of Molecular Biotechnology, Vienna BioCenter, 1030 Vienna, Austria
| | - Julius Brennecke
- Institute of Molecular Biotechnology, Vienna BioCenter, 1030 Vienna, Austria;
| | - Stefan L Ameres
- Institute of Molecular Biotechnology, Vienna BioCenter, 1030 Vienna, Austria;
- Max Perutz Labs, University of Vienna, Vienna BioCenter, 1030 Vienna, Austria
| | - Dinshaw J Patel
- Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065;
| |
Collapse
|
|
46
|
Maguire S, Lohman GJS, Guan S. A low-bias and sensitive small RNA library preparation method using randomized splint ligation. Nucleic Acids Res 2020; 48:e80. [PMID: 32496547 PMCID: PMC7641310 DOI: 10.1093/nar/gkaa480] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2020] [Revised: 04/22/2020] [Accepted: 05/27/2020] [Indexed: 12/16/2022] Open
Abstract
Small RNAs are important regulators of gene expression and are involved in human development and disease. Next generation sequencing (NGS) allows for scalable, genome-wide studies of small RNA; however, current methods are challenged by low sensitivity and high bias, limiting their ability to capture an accurate representation of the cellular small RNA population. Several studies have shown that this bias primarily arises during the ligation of single-strand adapters during library preparation, and that this ligation bias is magnified by 2′-O-methyl modifications (2′OMe) on the 3′ terminal nucleotide. In this study, we developed a novel library preparation process using randomized splint ligation with a cleavable adapter, a design which resolves previous challenges associated with this ligation strategy. We show that a randomized splint ligation based workflow can reduce bias and increase the sensitivity of small RNA sequencing for a wide variety of small RNAs, including microRNA (miRNA) and tRNA fragments as well as 2′OMe modified RNA, including Piwi-interacting RNA and plant miRNA. Finally, we demonstrate that this workflow detects more differentially expressed miRNA between tumorous and matched normal tissues. Overall, this library preparation process allows for highly accurate small RNA sequencing and will enable studies of 2′OMe modified RNA with new levels of detail.
Collapse
Affiliation(s)
- Sean Maguire
- New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA
| | | | - Shengxi Guan
- New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA
| |
Collapse
|
|
47
|
Grentzinger T, Oberlin S, Schott G, Handler D, Svozil J, Barragan-Borrero V, Humbert A, Duharcourt S, Brennecke J, Voinnet O. A universal method for the rapid isolation of all known classes of functional silencing small RNAs. Nucleic Acids Res 2020; 48:e79. [PMID: 32496553 PMCID: PMC7641303 DOI: 10.1093/nar/gkaa472] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2019] [Revised: 05/05/2020] [Accepted: 05/25/2020] [Indexed: 01/30/2023] Open
Abstract
Diverse classes of silencing small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Here, we have streamlined various embodiments of a Q-sepharose-based RISC-purification method that relies on conserved biochemical properties of all ARGONAUTEs. We show, in multiple benchmarking assays, that the resulting 15-min benchtop extraction procedure allows simultaneous purification of all known classes of RISC-associated sRNAs without prior knowledge of the samples-intrinsic ARGONAUTE repertoires. Optimized under a user-friendly format, the method – coined ‘TraPR’ for Trans-kingdom, rapid, affordable Purification of RISCs – operates irrespectively of the organism, tissue, cell type or bio-fluid of interest, and scales to minute amounts of input material. The method is highly suited for direct profiling of silencing sRNAs, with TraPR-generated sequencing libraries outperforming those obtained via gold-standard procedures that require immunoprecipitations and/or lengthy polyacrylamide gel-selection. TraPR considerably improves the quality and consistency of silencing sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots or mammalian plasma, and regardless of RNA contaminants or RNA degradation status of samples.
Collapse
Affiliation(s)
- Thomas Grentzinger
- Department of Biology, Swiss Federal Institute of Technology (ETH), Zürich, 8092, Switzerland
| | - Stefan Oberlin
- Department of Biology, Swiss Federal Institute of Technology (ETH), Zürich, 8092, Switzerland
| | - Gregory Schott
- Department of Biology, Swiss Federal Institute of Technology (ETH), Zürich, 8092, Switzerland
| | - Dominik Handler
- Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, 1030, Austria
| | - Julia Svozil
- Department of Biology, Swiss Federal Institute of Technology (ETH), Zürich, 8092, Switzerland
| | | | - Adeline Humbert
- Institut Jacques Monod, Université de Paris, CNRS, Paris, 75013, France
| | - Sandra Duharcourt
- Institut Jacques Monod, Université de Paris, CNRS, Paris, 75013, France
| | - Julius Brennecke
- Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, 1030, Austria
| | - Olivier Voinnet
- Department of Biology, Swiss Federal Institute of Technology (ETH), Zürich, 8092, Switzerland
| |
Collapse
|
|
48
|
Comparison of Reproducibility, Accuracy, Sensitivity, and Specificity of miRNA Quantification Platforms. Cell Rep 2020; 29:4212-4222.e5. [PMID: 31851944 PMCID: PMC7499898 DOI: 10.1016/j.celrep.2019.11.078] [Citation(s) in RCA: 68] [Impact Index Per Article: 13.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2019] [Revised: 10/17/2019] [Accepted: 11/19/2019] [Indexed: 01/08/2023] Open
Abstract
Given the increasing interest in their use as disease biomarkers, the establishment of reproducible, accurate, sensitive, and specific platforms for microRNA (miRNA) quantification in biofluids is of high priority. We compare four platforms for these characteristics: small RNA sequencing (RNA-seq), FirePlex, EdgeSeq, and nCounter. For a pool of synthetic miRNAs, coefficients of variation for technical replicates are lower for EdgeSeq (6.9%) and RNA-seq (8.2%) than for FirePlex (22.4%); nCounter replicates are not performed. Receiver operating characteristic analysis for distinguishing present versus absent miRNAs shows small RNA-seq (area under curve 0.99) is superior to EdgeSeq (0.97), nCounter (0.94), and FirePlex (0.81). Expected differences in expression of placenta-associated miRNAs in plasma from pregnant and non-pregnant women are observed with RNA-seq and EdgeSeq, but not FirePlex or nCounter. These results indicate that differences in performance among miRNA profiling platforms impact ability to detect biological differences among samples and thus their relative utility for research and clinical use. Using pools of synthetic RNA oligonucleotides and standardized extracellular RNA samples, Godoy et al. compare small RNA sequencing to three targeted miRNA quantification platforms to evaluate reproducibility, bias, specificity and sensitivity, and accuracy. Each platform has strengths and limitations important to consider for biomarker discovery, clinical validation, and broad clinical use.
Collapse
|
|
49
|
Heinicke F, Zhong X, Zucknick M, Breidenbach J, Sundaram AY, T. Flåm S, Leithaug M, Dalland M, Rayner S, Lie BA, Gilfillan GD. An extension to: Systematic assessment of commercially available low-input miRNA library preparation kits. RNA Biol 2020; 17:1284-1292. [PMID: 32436772 PMCID: PMC7549702 DOI: 10.1080/15476286.2020.1761081] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2020] [Revised: 03/18/2020] [Accepted: 04/22/2020] [Indexed: 11/12/2022] Open
Abstract
High-throughput sequencing has emerged as the favoured method to study microRNA (miRNA) expression, but biases introduced during library preparation have been reported. We recently compared the performance (sensitivity, reliability, titration response and differential expression) of six commercially-available kits on synthetic miRNAs and human RNA, where library preparation was performed by the vendors. We hereby supplement this study with data from two further commonly used kits (NEBNext, NEXTflex) whose manufacturers initially declined to participate. NEXTflex demonstrated the highest sensitivity, which may reflect its use of partially-randomized adapter sequences, but overall performance was lower than the QIAseq and TailorMix kits. NEBNext showed intermediate performance. We reaffirm that biases are kit specific, complicating the comparison of miRNA datasets generated using different kits.
Collapse
Affiliation(s)
- Fatima Heinicke
- Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway
| | - Xiangfu Zhong
- Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway
| | - Manuela Zucknick
- Department of Biostatistics, Oslo Centre for Biostatistics and Epidemiology, University of Oslo, Oslo, Norway
| | - Johannes Breidenbach
- National Forest Inventory, Norwegian Institute for Bioeconomy Research, Ås, Norway
| | - Arvind Y.M. Sundaram
- Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway
| | - Siri T. Flåm
- Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway
| | - Magnus Leithaug
- Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway
| | - Marianne Dalland
- Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway
| | - Simon Rayner
- Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway
| | - Benedicte A. Lie
- Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway
| | - Gregor D. Gilfillan
- Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway
| |
Collapse
|
|
50
|
Joilin G, Gray E, Thompson AG, Bobeva Y, Talbot K, Weishaupt J, Ludolph A, Malaspina A, Leigh PN, Newbury SF, Turner MR, Hafezparast M. Identification of a potential non-coding RNA biomarker signature for amyotrophic lateral sclerosis. Brain Commun 2020; 2:fcaa053. [PMID: 32613197 PMCID: PMC7329382 DOI: 10.1093/braincomms/fcaa053] [Citation(s) in RCA: 36] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Objective biomarkers for the clinically heterogeneous adult-onset neurodegenerative disorder amyotrophic lateral sclerosis are crucial to facilitate assessing emerging therapeutics and improve the diagnostic pathway in what is a clinically heterogeneous syndrome. With non-coding RNA transcripts including microRNA, piwi-RNA and transfer RNA present in human biofluids, we sought to identify whether non-coding RNA in serum could be biomarkers for amyotrophic lateral sclerosis. Serum samples from our Oxford Study for Biomarkers in motor neurone disease/amyotrophic lateral sclerosis discovery cohort of amyotrophic lateral sclerosis patients (n = 48), disease mimics (n = 16) and age- and sex-matched healthy controls (n = 24) were profiled for non-coding RNA expression using RNA-sequencing, which showed a wide range of non-coding RNA to be dysregulated. We confirmed significant alterations with reverse transcription-quantitative PCR in the expression of hsa-miR-16-5p, hsa-miR-21-5p, hsa-miR-92a-3p, hsa-piR-33151, TRV-AAC4-1.1 and TRA-AGC6-1.1. Furthermore, hsa-miR-206, a previously identified amyotrophic lateral sclerosis biomarker, showed a binary-like pattern of expression in our samples. Using the expression of these non-coding RNA, we were able to discriminate amyotrophic lateral sclerosis samples from healthy controls in our discovery cohort using a random forest analysis with 93.7% accuracy with promise in predicting progression rate of patients. Importantly, cross-validation of this novel signature using a new geographically distinct cohort of samples from the United Kingdom and Germany with both amyotrophic lateral sclerosis and control samples (n = 156) yielded an accuracy of 73.9%. The high prediction accuracy of this non-coding RNA-based biomarker signature, even across heterogeneous cohorts, demonstrates the strength of our approach as a novel platform to identify and stratify amyotrophic lateral sclerosis patients.
Collapse
Affiliation(s)
- Greig Joilin
- School of Life Sciences, University of Sussex, Falmer, Brighton, UK
| | - Elizabeth Gray
- Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK
| | | | - Yoana Bobeva
- Centre for Neuroscience and Trauma, Blizard Institute, Queen Mary University of London, London, UK
| | - Kevin Talbot
- Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK
| | | | - Albert Ludolph
- Department of Neurology, University of Ulm, Ulm, Germany
| | - Andrea Malaspina
- Centre for Neuroscience and Trauma, Blizard Institute, Queen Mary University of London, London, UK
| | - P Nigel Leigh
- Department of Neuroscience, Brighton and Sussex Medical School, University of Sussex, Falmer, Brighton, UK
| | - Sarah F Newbury
- Department of Clinical and Experimental Medicine, Brighton and Sussex Medical School, University of Sussex, Falmer, Brighton, UK
| | - Martin R Turner
- Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK
| | - Majid Hafezparast
- School of Life Sciences, University of Sussex, Falmer, Brighton, UK
- Correspondence to: Majid Hafezparast, School of Life Sciences, University of Sussex, Falmer Brighton, BN1 9QG, UK E-mail:
| |
Collapse
|