Chapter 10: Transcription and RNA Processing 10.1 Types of RNA
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Coupling of Spliceosome Complexity to Intron Diversity
bioRxiv preprint doi: https://doi.org/10.1101/2021.03.19.436190; this version posted March 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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. Coupling of spliceosome complexity to intron diversity Jade Sales-Lee1, Daniela S. Perry1, Bradley A. Bowser2, Jolene K. Diedrich3, Beiduo Rao1, Irene Beusch1, John R. Yates III3, Scott W. Roy4,6, and Hiten D. Madhani1,6,7 1Dept. of Biochemistry and Biophysics University of California – San Francisco San Francisco, CA 94158 2Dept. of Molecular and Cellular Biology University of California - Merced Merced, CA 95343 3Department of Molecular Medicine The Scripps Research Institute, La Jolla, CA 92037 4Dept. of Biology San Francisco State University San Francisco, CA 94132 5Chan-Zuckerberg Biohub San Francisco, CA 94158 6Corresponding authors: [email protected], [email protected] 7Lead Contact 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.19.436190; this version posted March 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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. SUMMARY We determined that over 40 spliceosomal proteins are conserved between many fungal species and humans but were lost during the evolution of S. cerevisiae, an intron-poor yeast with unusually rigid splicing signals. We analyzed null mutations in a subset of these factors, most of which had not been investigated previously, in the intron-rich yeast Cryptococcus neoformans. -
Nuclear Expression of a Group II Intron Is Consistent with Spliceosomal Intron Ancestry
Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Nuclear expression of a group II intron is consistent with spliceosomal intron ancestry Venkata R. Chalamcharla, M. Joan Curcio, and Marlene Belfort1 Center for Medical Sciences, Wadsworth Center, New York State Department of Health, Albany, New York 12208, USA; and School of Public Health, State University of New York at Albany, Albany, New York 12201, USA Group II introns are self-splicing RNAs found in eubacteria, archaea, and eukaryotic organelles. They are mechanistically similar to the metazoan nuclear spliceosomal introns; therefore, group II introns have been invoked as the progenitors of the eukaryotic pre-mRNA introns. However, the ability of group II introns to function outside of the bacteria-derived organelles is debatable, since they are not found in the nuclear genomes of eukaryotes. Here, we show that the Lactococcus lactis Ll.LtrB group II intron splices accurately and efficiently from different pre-mRNAs in a eukaryote, Saccharomyces cerevisiae. However, a pre-mRNA harboring a group II intron is spliced predominantly in the cytoplasm and is subject to nonsense-mediated mRNA decay (NMD), and the mature mRNA from which the group II intron is spliced is poorly translated. In contrast, a pre-mRNA bearing the Tetrahymena group I intron or the yeast spliceosomal ACT1 intron at the same location is not subject to NMD, and the mature mRNA is translated efficiently. Thus, a group II intron can splice from a nuclear transcript, but RNA instability and translation defects would have favored intron loss or evolution into protein-dependent spliceosomal introns, consistent with the bacterial group II intron ancestry hypothesis. -
Bacterial RNA Polymerase Can Retain Σ Throughout Transcription
Bacterial RNA polymerase can retain σ70 throughout transcription Timothy T. Hardena,b, Christopher D. Wellsc, Larry J. Friedmanb, Robert Landickd,e, Ann Hochschildc, Jane Kondeva,1, and Jeff Gellesb,1 aDepartment of Physics, Brandeis University, Waltham, MA 02454; bDepartment of Biochemistry, Brandeis University, Waltham, MA 02454; cDepartment of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115; dDepartment of Biochemistry, University of Wisconsin, Madison, WI 53706; and eDepartment of Bacteriology, University of Wisconsin, Madison, WI 53706 Edited by Jeffrey W. Roberts, Cornell University, Ithaca, NY, and approved December 10, 2015 (received for review July 15, 2015) Production of a messenger RNA proceeds through sequential stages early elongation pause. This early elongation pause, in turn, al- of transcription initiation and transcript elongation and termination. lows loading of an antitermination factor that enables tran- During each of these stages, RNA polymerase (RNAP) function is scription of the late gene operon (10, 13–15). Similar promoter- regulated by RNAP-associated protein factors. In bacteria, RNAP- proximal pause elements are also associated with many E. coli associated σ factors are strictly required for promoter recognition promoters (16–19), but the function of these elements is yet and have historically been regarded as dedicated initiation factors. unknown. Furthermore, σ70 interaction sites on core RNAP par- However, the primary σ factor in Escherichia coli, σ70,canremain tially overlap with those of transcription elongation factors such as associated with RNAP during the transition from initiation to elon- NusA, NusG, and RfaH (20–23). This and other evidence raises the gation, influencing events that occur after initiation. Quantitative possibility that σ70 retained in TECs sterically occludes the binding studies on the extent of σ70 retention have been limited to com- of other factors, which in turn could affect processes modulated by plexes halted during early elongation. -
High-Resolution RNA 3 -Ends Mapping of Bacterial Rho-Dependent
Nucleic Acids Research, 2018 1 doi: 10.1093/nar/gky274 High-resolution RNA 3-ends mapping of bacterial Rho-dependent transcripts Daniel Dar and Rotem Sorek* Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel Received February 21, 2018; Revised March 29, 2018; Editorial Decision March 31, 2018; Accepted April 04, 2018 ABSTRACT defined according to their dependence on the proteinaceous termination factor, Rho (4). Transcription termination in bacteria can occur ei- Rho-independent terminators, also known as intrinsic ther via Rho-dependent or independent (intrinsic) terminators, efficiently destabilize the elongating RNA- mechanisms. Intrinsic terminators are composed of polymerase (RNAP) complex in the absence of Rho and a stem-loop RNA structure followed by a uridine are encoded by a short ∼30nt DNA sequence downstream stretch and are known to terminate in a precise man- of the protein-coding region of the gene, as well as in some ner. In contrast, Rho-dependent terminators have regulatory 5 mRNA leaders (5,6). These terminators are more loosely defined characteristics and are thought composed of two main modules: a GC-rich sequence that to terminate in a diffuse manner. While transcripts folds into an energetically stable stem-loop RNA structure ending in an intrinsic terminator are protected from and a 7–8 nt uridine rich sequence (7–10). Termination ini- 3-5 exonuclease digestion due to the stem-loop tiates when the U-rich tract is transcribed and occupies the transcription bubble, causing the RNAP to pause and al- structure of the terminator, it remains unclear what lowing the upstream stem-loop forming sequence to nucle- protects Rho-dependent transcripts from being de- ate, which disrupts the transcription complex (8,9,11). -
Molecular Basis of the Function of Transcriptional Enhancers
cells Review Molecular Basis of the Function of Transcriptional Enhancers 1,2, 1, 1,3, Airat N. Ibragimov y, Oleg V. Bylino y and Yulii V. Shidlovskii * 1 Laboratory of Gene Expression Regulation in Development, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., 119334 Moscow, Russia; [email protected] (A.N.I.); [email protected] (O.V.B.) 2 Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., 119334 Moscow, Russia 3 I.M. Sechenov First Moscow State Medical University, 8, bldg. 2 Trubetskaya St., 119048 Moscow, Russia * Correspondence: [email protected]; Tel.: +7-4991354096 These authors contributed equally to this study. y Received: 30 May 2020; Accepted: 3 July 2020; Published: 5 July 2020 Abstract: Transcriptional enhancers are major genomic elements that control gene activity in eukaryotes. Recent studies provided deeper insight into the temporal and spatial organization of transcription in the nucleus, the role of non-coding RNAs in the process, and the epigenetic control of gene expression. Thus, multiple molecular details of enhancer functioning were revealed. Here, we describe the recent data and models of molecular organization of enhancer-driven transcription. Keywords: enhancer; promoter; chromatin; transcriptional bursting; transcription factories; enhancer RNA; epigenetic marks 1. Introduction Gene transcription is precisely organized in time and space. The process requires the participation of hundreds of molecules, which form an extensive interaction network. Substantial progress was achieved recently in our understanding of the molecular processes that take place in the cell nucleus (e.g., see [1–9]). -
Sequences at the Exon-Intron Boundaries* (Split Gene/Mrna Splicing/Eukaryotic Gene Structure) R
Proc. Nati. Acad. Sci. USA Vol. 75, No. 10, pp. 4853-4857, October 1978 Biochemistry Ovalbumin gene: Evidence for a leader sequence in mRNA and DNA sequences at the exon-intron boundaries* (split gene/mRNA splicing/eukaryotic gene structure) R. BREATHNACH, C. BENOIST, K. O'HARE, F. GANNON, AND P. CHAMBON Laboratoire de Genetique Mol6culaire des Eucaryotes du Centre National de la Recherche Scientifique, Unite 44 de l'Institut National de la Sant6 et de la Recherche MWdicale, Institut de Chimie Biologique, Facult6 de Melecine, Strasbourg 67085, France Communicated by A. Frey-Wyssling, July 31, 1978 ABSTRACT Selected regions of cloned EcoRI fragments the 5' end of ov-mRNA and have revealed some interesting of the chicken ovalbumin gene have been sequenced. The po- features in the DNA sequences at exon-intron boundaries. sitions where the sequences coding for ovalbumin mRNA (ov- mRNA) are interrupted in the genome have been determined, and a previously unreported interruption in the DNA sequences MATERIALS AND METHODS coding for the 5' nontranslated region of the messenger has been discovered. Because directly repeated sequences are found at Plasmid pCR1 ov 2.1 containing the ov-ds-cDNA insert was exon-intron boundaries, the nucleotide sequence alone cannot prepared as described (9). EcoRI fragments "b," "c," and "d" define unique excision-ligation points for the processing of a previously cloned in X vectors (3) were transferred to the plas- possible ov-mRNA precursor. However, the sequences in these mid pBR 322. An EcoRI/HindIII of the EcoRI boundary regions share common features; this leads to the fragment proposal that there are, in fact, unique excision-ligation points fragment "a" containing the entirety of exon 7 (Fig. -
RNA Polymerase Clamp Movement Aids Dissociation from DNA but Is Not Required for RNA Release at Intrinsic Terminators
bioRxiv preprint doi: https://doi.org/10.1101/453969; this version posted October 26, 2018. 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. RNA polymerase clamp movement aids dissociation from DNA but is not required for RNA release at intrinsic terminators Michael J Bellecourta, Ananya Ray-Sonia,1, Alex Harwiga, Rachel Anne Mooneya, Robert Landicka,b* Departments of aBiochemistry and bBacteriology, University of Wisconsin–Madison, Madison, WI 53706, USA 1Present address: Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA *To whom correspondence should be addressed; Tel: +1 (608) 265-8475; Fax: +1 (608) 890- 2415; E-mail: [email protected] Highlights • The contributions of RNAP conformation changes during termination are ill-defined • Restricting RNAP clamp movement affects elongation rate, but not termination rate • RNA but not DNA can release at terminators when clamp movement is restricted • Inhibiting TL conformational flexibility impairs both RNA and DNA release Keywords: Escherichia coli, Termination commitment, Transcription elongation, Transcription pausing, Trigger loop Abbreviations: EC, elongating transcription complex; fxlink, crosslinking efficiency; Cys-pair, cysteine-pair; MccJ25, microcin J25; NA, nucleic acid; RNAP, RNA polymerase; TE, termination (commitment) efficiency; Thp, terminator hairpin; TL, trigger loop Declarations of interest: none Bellecourt et al. Page 1 10/25/2018 bioRxiv preprint doi: https://doi.org/10.1101/453969; this version posted October 26, 2018. 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. -
Enhancer Rnas Are an Important Regulatory Layer of the Epigenome
REVIEW ARTICLE https://doi.org/10.1038/s41594-020-0446-0 Enhancer RNAs are an important regulatory layer of the epigenome Vittorio Sartorelli 1 and Shannon M. Lauberth 2 ✉ Noncoding RNAs (ncRNAs) direct a remarkable number of diverse functions in development and disease through their regula- tion of transcription, RNA processing and translation. Leading the charge in the RNA revolution is a class of ncRNAs that are synthesized at active enhancers, called enhancer RNAs (eRNAs). Here, we review recent insights into the biogenesis of eRNAs and the mechanisms underlying their multifaceted functions and consider how these findings could inform future investigations into enhancer transcription and eRNA function. he explosion of high-throughput sequencing data has Many different models for how enhancers function in gene con- revealed the complexity and diversity of the transcriptome. trol have been proposed since their initial discovery nearly four TThese data have also unexpectedly revealed that only 1–2% decades ago19–21. Specifically, there is considerable evidence demon- of the transcriptome provides instructions for the synthesis of strating that looping of distal enhancers to their target promoters is functional proteins, while the remaining 98–99% gives rise to a required for enhancer function (reviewed in ref. 22). For example, plethora of ncRNAs, including transfer RNAs (tRNAs), ribosomal a key study revealed that experimental induction of chromatin RNAs (rRNAs), intronic RNAs, small nuclear (sn)RNAs, small looping between the mouse β-globin (Hbb) promoter and its asso- nucleolar (sno)RNAs, microRNAs (miRNAs) and long noncoding ciated enhancer region results in transcriptional activation of the RNAs (lncRNAs). A recent addition to the expanding list of regu- Hbb gene23. -
The RNA Hairpin (Escherichia Coli/RNA Polymerase/Rho-Independent Termination) KEVIN S
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 8793-8797, September 1995 Biochemistry Transcription termination at intrinsic terminators: The role of the RNA hairpin (Escherichia coli/RNA polymerase/rho-independent termination) KEVIN S. WILSONt AND PETER H. VON HIPPEL Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403 Contributed by Peter H. von Hippel, April 28, 1995 ABSTRACT Intrinsic termination of transcription in protein-dependent antitermination (Rees et al., unpublished Escherichia coli involves the formation of an RNA hairpin in data) and is likely to be quite general. In this view transcript the nascent RNA. This hairpin plays a central role in the termination is considered to be possible, in principle, at every release of the transcript and polymerase at intrinsic termi- template position. In practice, however, termination does not nation sites on the DNA template. We have created variants of occur at most template positions because the stability of the the AtR2 terminator hairpin and examined the relationship elongation complex results in characteristic half-times for between the structure and stability of this hairpin and the complex dissociation and RNA release of hours or days, while template positions and efficiencies of termination. The results the average dwell-time for elongation at a given template were used to test the simple nucleic acid destabilization model position at saturating NTP concentrations is 10-50 msec (3). of Yager and von Hippel and showed that this model must be It has been shown for the AtR2 terminator (2) and confirmed modified to provide a distinct role for the rU-rich sequence in for the AtR' terminator (Rees et al., unpublished data) that the the nascent RNA, since a perfect palindromic sequence that is termination pathway becomes accessible at intrinsic termina- sufficiently long to form an RNA hairpin that could destabilize tors because the transcription complex is massively destabi- the entire putative 12-bp RNADNA hybrid does not trigger lized in the vicinity of these sites. -
Enhancer Rnas: Transcriptional Regulators and Workmates of Namirnas in Myogenesis
Odame et al. Cell Mol Biol Lett (2021) 26:4 https://doi.org/10.1186/s11658-021-00248-x Cellular & Molecular Biology Letters REVIEW Open Access Enhancer RNAs: transcriptional regulators and workmates of NamiRNAs in myogenesis Emmanuel Odame , Yuan Chen, Shuailong Zheng, Dinghui Dai, Bismark Kyei, Siyuan Zhan, Jiaxue Cao, Jiazhong Guo, Tao Zhong, Linjie Wang, Li Li* and Hongping Zhang* *Correspondence: [email protected]; zhp@sicau. Abstract edu.cn miRNAs are well known to be gene repressors. A newly identifed class of miRNAs Farm Animal Genetic Resources Exploration termed nuclear activating miRNAs (NamiRNAs), transcribed from miRNA loci that and Innovation Key exhibit enhancer features, promote gene expression via binding to the promoter and Laboratory of Sichuan enhancer marker regions of the target genes. Meanwhile, activated enhancers pro- Province, College of Animal Science and Technology, duce endogenous non-coding RNAs (named enhancer RNAs, eRNAs) to activate gene Sichuan Agricultural expression. During chromatin looping, transcribed eRNAs interact with NamiRNAs University, Chengdu 611130, through enhancer-promoter interaction to perform similar functions. Here, we review China the functional diferences and similarities between eRNAs and NamiRNAs in myogen- esis and disease. We also propose models demonstrating their mutual mechanism and function. We conclude that eRNAs are active molecules, transcriptional regulators, and partners of NamiRNAs, rather than mere RNAs produced during enhancer activation. Keywords: Enhancer RNA, NamiRNAs, MicroRNA, Myogenesis, Transcriptional regulator Introduction Te identifcation of lin-4 miRNA in Caenorhabditis elegans in 1993 [1] triggered research to discover and understand small microRNAs’ (miRNAs) mechanisms. Recently, some miRNAs are reported to activate target genes during transcription via base pairing to the 3ʹ or 5ʹ untranslated regions (3ʹ or 5ʹ UTRs), the promoter [2], and the enhancer regions [3]. -
The Lifespan of an MLL-Rearranged Therapy-Related Paediatric AML
Bone Marrow Transplantation (2015) 50, 1382–1384 © 2015 Macmillan Publishers Limited All rights reserved 0268-3369/15 www.nature.com/bmt LETTER TO THE EDITOR From initiation to eradication: the lifespan of an MLL-rearranged therapy-related paediatric AML Bone Marrow Transplantation (2015) 50, 1382–1384; doi:10.1038/ amplified by RT-PCR. DCP1A is predominantly cytoplasmic as part bmt.2015.155; published online 6 July 2015 of a protein complex involved in degradation of mRNAs.8 Upon stimulation by TGFB1 and interaction with SMAD4, it becomes nuclear contributing to the transactivation of TGF-α target genes.9 Therapy-related AML (tAML) is one of the most prevalent DCP1A is a novel MLL fusion partner in AML. The in vitro secondary malignant neoplasms in paediatric cancer survivors. transforming capacity of MLL-DCP1A was weaker than the more Estimates of incidence and association with causative chemo- common MLL-ENL fusion but it could nevertheless be clearly therapeutic agents are mainly based on epidemiological data. verified by colony formation in retroviral transduction-replating 10 Individual courses revealing the exact timing of the first clone assays (Figure 1d). appearance and its development under treatment are limited The cumulative sampling of bone marrow during preceding owing to the overall low incidence, which does not justify neuroblastoma treatment offered the unique possibility to repeated bone marrow sampling in order to detect the occurrence quantitatively backtrack the origin of the tAML by quantitative of potentially leukemic cells. real-time PCR using the genomic MLL-DCP1A breakpoint as a Here we report a unique case of tAML arising after neuro- molecular marker. -
Spliceosomal Intronogenesis
Spliceosomal intronogenesis Sujin Leea and Scott W. Stevensb,c,1 aGraduate Program in Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712; bDepartment of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712; and cInstitute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712 Edited by Jef D. Boeke, New York University School of Medicine, New York, NY, and approved April 20, 2016 (received for review March 30, 2016) The presence of intervening sequences, termed introns, is a defining it is clear that introns massively infiltrated the genome of the last characteristic of eukaryotic nuclear genomes. Once transcribed into eukaryotic common ancestor and that introns have continued to be pre-mRNA, these introns must be removed within the spliceosome gained and lost over evolutionary time (11, 18). before export of the processed mRNA to the cytoplasm, where it is Here we report the use of a reporter system designed to detect translated into protein. Although intron loss has been demonstrated events of intron gain and intron loss in the budding yeast Sac- experimentally, several mysteries remain regarding the origin and charomyces cerevisiae. With this reporter, we have captured, to our propagation of introns. Indeed, documented evidence of gain of an knowledge, the first verified examples of intron gain via intron intron has only been suggested by phylogenetic analyses. We report transposition in any eukaryote. the use of a strategy that detects selected intron gain and loss events. We have experimentally verified, to our knowledge, the first demon- Results strations of intron transposition in any organism.