Small RNA-Sequencing: Approaches and Considerations for Mirna Analysis
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Comparison of the Effects on Mrna and Mirna Stability Arian Aryani and Bernd Denecke*
Aryani and Denecke BMC Research Notes (2015) 8:164 DOI 10.1186/s13104-015-1114-z RESEARCH ARTICLE Open Access In vitro application of ribonucleases: comparison of the effects on mRNA and miRNA stability Arian Aryani and Bernd Denecke* Abstract Background: MicroRNA has become important in a wide range of research interests. Due to the increasing number of known microRNAs, these molecules are likely to be increasingly seen as a new class of biomarkers. This is driven by the fact that microRNAs are relatively stable when circulating in the plasma. Despite extensive analysis of mechanisms involved in microRNA processing, relatively little is known about the in vitro decay of microRNAs under defined conditions or about the relative stabilities of mRNAs and microRNAs. Methods: In this in vitro study, equal amounts of total RNA of identical RNA pools were treated with different ribonucleases under defined conditions. Degradation of total RNA was assessed using microfluidic analysis mainly based on ribosomal RNA. To evaluate the influence of the specific RNases on the different classes of RNA (ribosomal RNA, mRNA, miRNA) ribosomal RNA as well as a pattern of specific mRNAs and miRNAs was quantified using RT-qPCR assays. By comparison to the untreated control sample the ribonuclease-specific degradation grade depending on the RNA class was determined. Results: In the present in vitro study we have investigated the stabilities of mRNA and microRNA with respect to the influence of ribonucleases used in laboratory practice. Total RNA was treated with specific ribonucleases and the decay of different kinds of RNA was analysed by RT-qPCR and miniaturized gel electrophoresis. -
Human 28S Rrna 5' Terminal Derived Small RNA Inhibits Ribosomal Protein Mrna Levels Shuai Li 1* 1. Department of Breast Cancer
bioRxiv preprint doi: https://doi.org/10.1101/618520; this version posted April 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Human 28s rRNA 5’ terminal derived small RNA inhibits ribosomal protein mRNA levels Shuai Li 1* 1. Department of Breast Cancer Pathology and Research Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin; Tianjin’s Clinical Research Center for Cancer, Tianjin 300060, China. * To whom correspondence should be addressed. Tel.: +86 22 23340123; Fax: +86 22 23340123; Email: [email protected] & [email protected]. bioRxiv preprint doi: https://doi.org/10.1101/618520; this version posted April 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Recent small RNA (sRNA) high-throughput sequencing studies reveal ribosomal RNAs (rRNAs) as major resources of sRNA. By reanalyzing sRNA sequencing datasets from Gene Expression Omnibus (GEO), we identify 28s rRNA 5’ terminal derived sRNA (named 28s5-rtsRNA) as the most abundant rRNA-derived sRNAs. These 28s5-rtsRNAs show a length dynamics with identical 5’ end and different 3’ end. Through exploring sRNA sequencing datasets of different human tissues, 28s5-rtsRNA is found to be highly expressed in bladder, macrophage and skin. We also show 28s5-rtsRNA is independent of microRNA biogenesis pathway and not associated with Argonaut proteins. Overexpression of 28s5-rtsRNA could alter the 28s/18s rRNA ratio and decrease multiple ribosomal protein mRNA levels. -
Global Analysis of Small RNA and Mrna Targets of Hfq
Blackwell Science, LtdOxford, UKMMIMolecular Microbiology 1365-2958Blackwell Publishing Ltd, 200350411111124Original ArticleA. Zhang et al.Global analysis of Hfq targets Molecular Microbiology (2003) 50(4), 1111–1124 doi:10.1046/j.1365-2958.2003.03734.x Global analysis of small RNA and mRNA targets of Hfq Aixia Zhang,1 Karen M. Wassarman,2 greatly expanded over the past few years (reviewed in Carsten Rosenow,3 Brian C. Tjaden,4† Gisela Storz1* Gottesman, 2002; Grosshans and Slack, 2002; Storz, and Susan Gottesman5* 2002; Wassarman, 2002; Massé et al., 2003). A subset of 1Cell Biology and Metabolism Branch, National Institute of these small RNAs act via short, interrupted basepairing Child Health and Development, Bethesda MD 20892, interactions with target mRNAs. How do these small USA. RNAs find and anneal to their targets? In Escherichia coli, 2Department of Bacteriology, University of Wisconsin, at least part of the answer lies in their association with Madison, WI 53706, USA. and dependence upon the RNA chaperone, Hfq. The 3Affymetrix, Santa Clara, CA 95051, USA. abundant Hfq protein was identified originally as a host 4Department of Computer Science, University of factor for RNA phage Qb replication (Franze de Fernan- Washington, Seattle, WA 98195, USA. dez et al., 1968), but later hfq mutants were found to 5Laboratory of Molecular Biology, National Cancer exhibit multiple phenotypes (Brown and Elliott, 1996; Muf- Institute, Bethesda, MD 20892, USA. fler et al., 1996). These defects are, at least in part, a reflection of the fact that Hfq is required for the function of several small RNAs including DsrA, RprA, Spot42, Summary OxyS and RyhB (Zhang et al., 1998; Sledjeski et al., Hfq, a bacterial member of the Sm family of RNA- 2001; Massé and Gottesman, 2002; Møller et al., 2002). -
U6 Small Nuclear RNA Is Transcribed by RNA Polymerase III (Cloned Human U6 Gene/"TATA Box"/Intragenic Promoter/A-Amanitin/La Antigen) GARY R
Proc. Nati. Acad. Sci. USA Vol. 83, pp. 8575-8579, November 1986 Biochemistry U6 small nuclear RNA is transcribed by RNA polymerase III (cloned human U6 gene/"TATA box"/intragenic promoter/a-amanitin/La antigen) GARY R. KUNKEL*, ROBIN L. MASERt, JAMES P. CALVETt, AND THORU PEDERSON* *Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545; and tDepartment of Biochemistry, University of Kansas Medical Center, Kansas City, KS 66103 Communicated by Aaron J. Shatkin, August 7, 1986 ABSTRACT A DNA fragment homologous to U6 small 4A (20) was screened with a '251-labeled U6 RNA probe (21, nuclear RNA was isolated from a human genomic library and 22) using a modified in situ plaque hybridization protocol sequenced. The immediate 5'-flanking region of the U6 DNA (23). One of several positive clones was plaque-purified and clone had significant homology with a potential mouse U6 gene, subsequently shown by restriction mapping to contain a including a "TATA box" at a position 26-29 nucleotides 12-kilobase-pair (kbp) insert. A 3.7-kbp EcoRI fragment upstream from the transcription start site. Although this containing U6-hybridizing sequences was subcloned into sequence element is characteristic of RNA polymerase II pBR322 for further restriction mapping. An 800-base-pair promoters, the U6 gene also contained a polymerase III "box (bp) DNA fragment containing U6 homologous sequences A" intragenic control region and a typical run of five thymines was excised using Ava I and inserted into the Sma I site of at the 3' terminus (noncoding strand). The human U6 DNA M13mp8 replicative form DNA (M13/U6) (24). -
Small Non-Coding Rnaome of Ageing Chondrocytes
International Journal of Molecular Sciences Article Small Non-Coding RNAome of Ageing Chondrocytes Panagiotis Balaskas 1,* , Jonathan A. Green 2, Tariq M. Haqqi 2, Philip Dyer 1, Yalda A. Kharaz 1, Yongxiang Fang 3 , Xuan Liu 3, Tim J.M. Welting 4 and Mandy J. Peffers 1,* 1 Institute of Life Course and Medical Sciences, William Henry Duncan Building, 6 West Derby Street, Liverpool L7 8TX, UK; [email protected] (P.D.); [email protected] (Y.A.K.) 2 Department of Anatomy and Neurobiology, Northeast Ohio Medical University, Rootstown, OH 44272, USA; [email protected] (J.A.G.); [email protected] (T.M.H.) 3 Centre for Genomic Research, Institute of Integrative Biology, Biosciences Building, Crown Street, University of Liverpool, Liverpool L69 7ZB, UK; [email protected] (Y.F.); [email protected] (X.L.) 4 Department of Orthopaedic Surgery, Maastricht University Medical Centre, 6202 AZ Maastricht, The Netherlands; [email protected] * Correspondence: [email protected] (P.B.); peff[email protected] (M.J.P.); Tel.: +44-0787-269-2102 (M.J.P.) Received: 20 July 2020; Accepted: 4 August 2020; Published: 7 August 2020 Abstract: Ageing is a leading risk factor predisposing cartilage to osteoarthritis. However, little research has been conducted on the effect of ageing on the expression of small non-coding RNAs (sncRNAs). RNA from young and old chondrocytes from macroscopically normal equine metacarpophalangeal joints was extracted and subjected to small RNA sequencing (RNA-seq). Differential expression analysis was performed in R using package DESeq2. For transfer RNA (tRNA) fragment analysis, tRNA reads were aligned to horse tRNA sequences using Bowtie2 version 2.2.5. -
Plant Non-Coding Rnas: Origin, Biogenesis, Mode of Action and Their Roles in Abiotic Stress
International Journal of Molecular Sciences Review Plant Non-Coding RNAs: Origin, Biogenesis, Mode of Action and Their Roles in Abiotic Stress Joram Kiriga Waititu 1, Chunyi Zhang 1, Jun Liu 2 and Huan Wang 1,* 1 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China; [email protected] (J.K.W.); [email protected] (C.Z.) 2 National Key Facility for Crop Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China; [email protected] * Correspondence: [email protected] Received: 13 October 2020; Accepted: 4 November 2020; Published: 9 November 2020 Abstract: As sessile species, plants have to deal with the rapidly changing environment. In response to these environmental conditions, plants employ a plethora of response mechanisms that provide broad phenotypic plasticity to allow the fine-tuning of the external cues related reactions. Molecular biology has been transformed by the major breakthroughs in high-throughput transcriptome sequencing and expression analysis using next-generation sequencing (NGS) technologies. These innovations have provided substantial progress in the identification of genomic regions as well as underlying basis influencing transcriptional and post-transcriptional regulation of abiotic stress response. Non-coding RNAs (ncRNAs), particularly microRNAs (miRNAs), short interfering RNAs (siRNAs), and long non-coding RNAs (lncRNAs), have emerged as essential regulators of plants abiotic stress response. However, shared traits in the biogenesis of ncRNAs and the coordinated cross-talk among ncRNAs mechanisms contribute to the complexity of these molecules and might play an essential part in regulating stress responses. Herein, we highlight the current knowledge of plant microRNAs, siRNAs, and lncRNAs, focusing on their origin, biogenesis, modes of action, and fundamental roles in plant response to abiotic stresses. -
A Small Nucleolar RNA Is Processed from an Intron of the Human Gene Encoding Ribosomal Protein $3
Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press A small nucleolar RNA is processed from an intron of the human gene encoding ribosomal protein $3 Kazimierz Tyc Tycowski, Mei-Di Shu, and Joan A. Steitz Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536-0812 USA A human small nucleolar RNA, identified previously in HeLa cells by anti-fibrillarin autoantibody precipitation and termed RNA X, has been characterized. It comprises two uridine-rich variants (148 and 146 nucleotides}, which we refer to as snRNA U15A and U15B. Secondary structure models predict for both variants a U15-specific stem-loop structure, as well as a new structural motif that contains conserved sequences and can also be recognized in the other fibrillarin-associated nucleolar snRNAs, U3, U14, and RNA Y. The single-copy gene for human U15A has been found unexpectedly to reside in intron 1 of the ribosomal protein $3 gene; the U15A sequence appears on the same strand as the $3 mRNA and does not exhibit canonical transcription signals for nuclear RNA polymerases. U15A RNA is processed in vitro from $3 intron 1 transcripts to yield the correct 5' end with a 5'-monophosphate; the in vitro system requires ATP for 3' cleavage, which occurs a few nucleotides downstream of the mature end. The production of a single primary transcript specifying the mRNA for a ribosomal or nucleolar protein and a nucleolar snRNA may constitute a general mechanism for balancing the levels of nucleolar components in vertebrate cells. -
Validation of a Core RNA-Interference Pathway As a Tool for Reverse Genetics in the Emergent Model Phototrophic Endosymbiosis, Paramecium Bursaria
bioRxiv preprint doi: https://doi.org/10.1101/2020.12.16.423098; this version posted December 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Validation of a Core RNA-interference Pathway as a Tool for Reverse Genetics in the Emergent Model Phototrophic Endosymbiosis, Paramecium bursaria Benjamin H. Jenkins1,2*, Finlay Maguire3*, Guy Leonard1,2, Joshua D. Eaton1, Steven West1, Benjamin E. Housden1, David, S. Milner1,2, & Thomas A. Richards1,2* 1Living Systems Institute and Biosciences, University of Exeter, Devon EX4 4QD, UK 2Department of Zoology, University of Oxford, 11a Mansfield Road, Oxford OX1 3SZ, UK 3Faculty of Computer Science, Dalhousie University, 6050 University Ave, Halifax NS B3H 1W5, Canada *Correspondence: [email protected] & [email protected] KEYWORDS Paramecium bursaria, algae, symbiosis, RNAi, Dicer, Argonaute, Pds1, siRNA ABSTRACT Endosymbiosis was fundamental for the evolution of eukaryotic cellular complexity. The genetic basis of an emergent endosymbiotic interaction may be investigated using forward and reverse genetic techniques, such as RNA-interference (RNAi). However, distinguishing small (s)RNA processing pathways in a eukaryote-eukaryote endosymbiotic interaction is complex. Here, we investigate the repertoire of candidate RNAi pathway protein-encoding genes in the model nascent endosymbiotic system, Paramecium bursaria–Chlorella spp. Using comparative genomics and transcriptomics supported by phylogenetics, we identify putative essential proteome components of the small interfering (si)RNA, scan (scn)RNA, and internal eliminated sequence (ies)RNA pathways encoded by P. -
Small-RNA Sequencing Libraries with Greatly Reduced Adaptor-Dimer Background
ADVERTISING fEATURE APPLICATION NOTES Small-RNA sequencing libraries with greatly reduced adaptor-dimer background Conventional methods for preparing small-RNA–seq libraries by adaptor ligation generate a significant amount of adaptor dimer, thereby resulting in wasted sequencing reads. These methods also do not capture small 5’-capped and 5’-triphosphorylated RNAs. The ScriptMiner™ small-RNA–seq library preparation technology overcomes these limitations. ScriptMiner™ small-RNA libraries contain greatly reduced amounts of adaptor-dimer when compared to conventional methods and also generate coverage that is characteristic of the entire small-RNA transcriptome. The small-RNA transcriptome contains a diverse array of RNAs, such 3’-adaptor oligonucleotide, which can form undesired adaptor-dimers, as microRNA (miRNA), small nucleolar RNA (snoRNA), small nuclear is then enzymatically removed. The default procedure provides the RNA (snRNA), small interfering RNA (siRNA) and piwi-interacting RNA option to tag only 5’ monophosphorylated RNAs, such as miRNA. (piRNA). The biogenesis of these classes of RNAs results in a variety An alternative step—treatment with tobacco acid pyrophosphatase of different modifications at the 5’ end: 5’-monophosphate (5’ pN), 5’-triphosphate (5’pppN) or 5’-cap (GpppN). In addition, the 3’ end of Small RNA, RNA fragments with or without a 5´ cap the RNA may be modified to include a 2’-O-methyl, 3’-OH instead of 5´ Gppp OH 5´ p OH 5´ p OH the normal 2’,3’-OH. 5´ app x Ligate 3´ adaptor Current methods of preparing small-RNA–seq libraries involve ligation-tagging (adaptor-ligation) of the 3’ and 5’ ends of the RNA, 5´ Gppp/ppp/p x 5´ app x Nature America, Inc. -
Emerging Data on the Diversity of Molecular Mechanisms Involving C/D Snornas
non-coding RNA Review Emerging Data on the Diversity of Molecular Mechanisms Involving C/D snoRNAs Laeya Baldini, Bruno Charpentier * and Stéphane Labialle * Campus Brabois Santé, Université de Lorraine, CNRS, IMoPA, F-54000 Nancy, France; [email protected] * Correspondence: [email protected] (B.C.); [email protected] (S.L.); Tel.: +33-3-72-74-66-27 (B.C.); +33-3-72-74-66-51 (S.L.) Abstract: Box C/D small nucleolar RNAs (C/D snoRNAs) represent an ancient family of small non-coding RNAs that are classically viewed as housekeeping guides for the 20-O-methylation of ribosomal RNA in Archaea and Eukaryotes. However, an extensive set of studies now argues that they are involved in mechanisms that go well beyond this function. Here, we present these pieces of evidence in light of the current comprehension of the molecular mechanisms that control C/D snoRNA expression and function. From this inventory emerges that an accurate description of these activities at a molecular level is required to let the snoRNA field enter in a second age of maturity. Keywords: box C/D small nucleolar RNA; C/D snoRNA; small nucleolar ribonucleoprotein; snoRNP; C/D snoRNP biogenesis; nucleolus; molecular biology 1. Introduction Citation: Baldini, L.; Charpentier, B.; On several occasions, the fortuitous identification of RNAs with odd features turned Labialle, S. Emerging Data on the out to be at the origin of a new family of non-coding RNAs (ncRNAs). A perfect example is Diversity of Molecular Mechanisms the characterization of development timing mutants in C. -
Specialization and Evolution of Endogenous Small RNA Pathways
REVIEWS Specialization and evolution of endogenous small RNA pathways Elisabeth J. Chapman* and James C. Carrington‡ Abstract | The specificity of RNA silencing is conferred by small RNA guides that are processed from structured RNA or dsRNA. The core components for small RNA biogenesis and effector functions have proliferated and specialized in eukaryotic lineages, resulting in diversified pathways that control expression of endogenous and exogenous genes, invasive elements and viruses, and repeated sequences. Deployment of small RNA pathways for spatiotemporal regulation of the transcriptome has shaped the evolution of eukaryotic genomes and contributed to the complexity of multicellular organisms. Genome expansion in higher plants, animals and other and genome-wide deployment of silencing mechanisms multicellular eukaryotes is partially attributed to an is high, it also includes a broader perspective from other increased capacity for the spatiotemporal control of systems. We focus primarily on endogenous regulatory gene expression that is required for development1,2. pathways, with only brief discussions of antiviral silenc- Throughout evolution, protein components of transcrip- ing mechanisms in cases in which they are of particular tional, post-transcriptional and post-translational regula- relevance to the central topic of this Review. tory pathways have proliferated, expanding the potential for regulatory specificity. Proteins that are involved in Diversity of small RNA pathways transcription, for example, represent as much as 10% Early studies of transgene-induced RNA-silencing of the coding sequence in the genomes of metazoans1. phenomena, particularly in plants, revealed puzzling By contrast, specificity for RNA-mediated silencing- differences in the molecular and epigenetic properties based regulation is conferred by small RNA guides (for that are associated with silencing in distinct individuals3. -
Noncoding Rnas and Cancer January 8-11, 2012 • Eden Roc Renaissance Beach Resort and Spa • Miami Beach, FL
An AACR Special Conference Noncoding RNAs and Cancer January 8-11, 2012 • Eden Roc Renaissance Beach Resort and Spa • Miami Beach, FL Conference Chairperson Phillip A. Sharp David H. Koch Institute for Integrative Cancer Research at MIT Cambridge, MA Conference Co-Chairpersons Carlo M. Croce The Ohio State University Comprehensive Cancer Center Columbus, OH William C. Hahn Dana-Farber Cancer Institute Boston, MA Jeannie T. Lee Massachusetts General Hospital Boston, MA Eric N. Olson University of Texas Southwestern Medical Center Dallas, TX Table of Contents Supporters 3 Awards 4 Conference Program 7 Proceedings Invited Presentations 13 Proffered Presentations 25 Poster Session A 29 Poster Session B 51 Participant List 69 Author Index 77 Subject Index 83 PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 1 2 NONCODING RNAS AND CANCER The AACR thanks the following organizations for their generous support of this Noncoding RNAs and Cancer conference (as of 12/6/11). Major Supporter Supporter The AACR thanks the following organizations for their generous support of the travel awards provided at this conference. A complete list of awardees and their affiliations is available on pages 4 and 5. Award Supporters PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 3 Scholar-in-Training Awards Four presenters of meritorious abstracts have been selected by the Conference Co- Chairpersons to receive awards to attend this conference. All graduate and medical students, postdoctoral fellows, and physicians-in-training were eligible for consideration. The names of the Scholar-in-Training awardees, their affiliations, and their poster or proffered presentation numbers are provided below.