Post Transcriptional Modification Snurps
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A Protein Required for RNA Processing and Splicing in Neurospora
Proc. Nati. Acad. Sci. USA Vol. 89, pp. 1676-1680, March 1992 Genetics A protein required for RNA processing and splicing in Neurospora mitochondria is related to gene products involved in cell cycle protein phosphatase functions BEATRICE TURCQ*, KATHERINE F. DOBINSONt, NOBUFUSA SERIZAWAt, AND ALAN M. LAMBOWITZ§ Departments of Molecular Genetics and Biochemistry, and the Biotechnology Center, The Ohio State University, 484 West Twelfth Avenue, Columbus, OH 43210 Communicated by Thomas R. Cech, November 25, 1991 ABSTRACT The Neurospora crassa cyt4 mutants have specifically affect the splicing of group I introns and have pleiotropic defects in mitochondrial RNA splicing, 5' and 3' end relatively little effect on other mitochondrial RNA processing processing, and RNA turnover. Here, we show that the cyt-4 reactions (1, 4, 7). gene encodes a 120-kDa protein with significant similarity to By contrast, the cyt4 mutants have a complex phenotype the SSD1/SRK1 protein of Saceharomyces cerevisiae and the with defects in a number of different aspects of mitochondrial DIS3 protein of Sclhizosaccharomyces pombe, which have been RNA metabolism in addition to RNA splicing (9, 10). The five implicated in protein phosphatase functions that regulate cell cyt4 mutants are cold sensitive; they grow slowly at 250C and cycle and mitotic chromosome segregation. The CYT-4 protein more rapidly at 370C. Initial studies focusing on the mitochon- is present in mitochondria and is truncated or deficient in two drial large rRNA showed that the cyt4 mutants are defective cyt4 mutants. Assuming that the CYT-4 protein functions in a in both splicing and 3' end synthesis and that the splicing defect manner similar to the SSD1/SRK1 and DIS3 proteins, we infer is more pronounced at lower temperatures (9). -
Transcription to RNA from Gene to Phenotype
11/8/11 Transcription to RNA From Gene to Phenotype DNA Gene 2 • The central dogma: molecule – DNA->RNA->protein Gene 1 – One Gene, One Enzyme Gene 3 • Beadle & Tatum expts. • Transcription DNA strand 3! 5! – Initiation (template) A C C A A A C C G A G T – Elongation – Termination TRANSCRIPTION U G G U U U G G C U C A • mRNA processing mRNA 5! 3! – Introns and exons Codon TRANSLATION • Other types of RNA 11/9/2011 Protein Trp Phe Gly Ser Amino acid In Prokaryotes transcription and translation occur simultaneously In Eukaryotes Nuclear – Transcription and envelope translation occur in separate compartments TRANSCRIPTION DNA DNA TRANSCRIPTION of the cell Pre-mRNA RNA PROCESSING mRNA Ribosome – RNA transcripts are mRNA TRANSLATION modified before becoming true mRNA Ribosome Polypeptide TRANSLATION Polypeptide Figure 17.3a Figure 17.3b One Gene -> One Enzyme “One Gene -> One Enzyme” EXPERIMENT Class I Class II Class III Wild type Mutants Mutants Mutants Minimal Normal bread-mold cells can medium synthesize arginine from precursors (MM) (control) in the minimal medium MM + Ornithine Mutant 2 could MM + grow if either Citrulline Precursor Ornithine Citrulline Arginine citruline or MM + arginine was Specific enzymes (arrows) Arginine is an essential Arginine amino acid, required for (control) supplied. catalyze each step growth Therefore it must lack the enzyme to make Citruline Precursor Ornithine Citrulline Arginine 1 11/8/11 From Gene to Phenotype RNA Polymerase Non-template strand of DNA DNA Gene 2 RNA nucleotides molecule Gene 1 Gene 3 T C C A A A T 3 C T U ! 3 end DNA strand 3! 5! ! G (template) A C C A A A C C G A G T T A U G G A 5 C A U C C A C TRANSCRIPTION ! A T A A G G T T U G G U U U G G C U C A mRNA 5! 3! Direction of transcription 5! Codon (“downstream) Template TRANSLATION strand of DNA Protein Trp Phe Gly Ser New RNA Amino acid Synthesis of an RNA Transcript DNA is copied to make messenger RNA Promoter Transcription unit 5! 3! 3! 5! RNA polymerase DNA This is the “non-template” Start point binds to a promoter RNA polymerase strand. -
The Differential Interaction of Snrnps with Pre-Mrna Reveals Splicing Kinetics in Living Cells
Published October 4, 2010 This article has original data in the JCB Data Viewer JCB: Article http://jcb-dataviewer.rupress.org/jcb/browse/3011 The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells Martina Huranová,1 Ivan Ivani,1 Aleš Benda,2 Ina Poser,3 Yehuda Brody,4,5 Martin Hof,2 Yaron Shav-Tal,4,5 Karla M. Neugebauer,3 and David StanČk1 1Institute of Molecular Genetics and 2J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic 3Max Planck Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany 4The Mina and Everard Goodman Faculty of Life Sciences and 5Institute for Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan 52900, Israel recursor messenger RNA (pre-mRNA) splicing is Core components of the spliceosome, U2 and U5 snRNPs, catalyzed by the spliceosome, a large ribonucleo- associated with pre-mRNA for 15–30 s, indicating that protein (RNP) complex composed of five small nuclear splicing is accomplished within this time period. Additionally, P Downloaded from RNP particles (snRNPs) and additional proteins. Using live binding of U1 and U4/U6 snRNPs with pre-mRNA oc- cell imaging of GFP-tagged snRNP components expressed curred within seconds, indicating that the interaction of at endogenous levels, we examined how the spliceosome individual snRNPs with pre-mRNA is distinct. These results assembles in vivo. A comprehensive analysis of snRNP are consistent with the predictions of the step-wise model dynamics in the cell nucleus enabled us to determine of spliceosome assembly and provide an estimate on the snRNP diffusion throughout the nucleoplasm as well as rate of splicing in human cells. -
POLYADENYLATION REGULATION of U1A Mrna: CHARACTERIZING
STUDIES OF POLYADENYLATION REGULATION OF U1A mRNA BY AN RNP COMPLEX CONTAINING U1A AND U1 snRNP By ROSE MARIE CARATOZZOLO A dissertation submitted to the Graduate School – New Brunswick Rutgers, The State University of New Jersey and The Graduate School of Biomedical Sciences University of Medicine and Dentistry of New Jersey In partial fulfillment of the requirements for the degree of Doctor of Philosophy Graduate Program in Biochemistry Written under the direction of Samuel I. Gunderson, Ph.D., And approved by _____________________________ _____________________________ _____________________________ _____________________________ New Brunswick, New Jersey January, 2011 ABSTRACT OF THE DISSERTATION STUDIES OF POLYADENYLATION REGULATION OF U1A mRNA BY AN RNP COMPLEX CONTAINING U1A AND U1 snRNP By Rose Marie Caratozzolo Dissertation Director: Samuel I. Gunderson, Ph.D. The 3’-end processing of nearly all eukaryotic pre-mRNAs comprises multiple steps which culminate in the addition of a poly(A) tail, which is essential for mRNA stability, translation, and export. Consequently, polyadenylation regulation is an important component of gene expression. One way to regulate polyadenylation is to inhibit the activity of a single poly(A) site, as exemplified by the U1A protein that negatively autoregulates itself by binding to a Polyadenylation Inhibitory Element (PIE) site within the 3’ UTR of its own pre-mRNA. U1 snRNP, which is primarily involved in splice site recognition, inhibits poly(A) site activity in papillomaviruses by binding to 5’ splice site-like sequences, which have recently been named “U1-sites”. Here, a recently identified U1-site in the human U1A 3'UTR is examined and shown to synergize with the adjacent PIE site to inhibit polyadenylation. -
1 Figure 1. Electron Micrograph of a Three- Part Hybrid Showing The
The Journey to RNA Splicing and the Spliceosome Phillip A. Sharp, Institute Professor, Department of Biology and the Koch Institute for Integrative Cancer Research at MIT In keeping with the objective of this meeting “Forty Years of mRNA Splicing: From Discovery to Therapeutics” to provide historical perspective, I have outlined recollections of some of the events surrounding the discovery of RNA splicing. The focus will be on contributions from the MIT group. My account is clearly self-serving and omits many other, highly important, contributions to the field; hopefully, others will add their recollections of events to the meeting’s web site. THE TRANSITION TO MOLECULAR AND CELL BIOLOGY. During the last year of my graduate studies under Victor Bloomfield at the University of Illinois, I read the annual Cold Spring Harbor Symposium volume of 1966 with the title The Genetic Code where the structures of chromosomes were reviewed (1). At the time, I faced two major steps in my career, writing a thesis about the physical chemistry of stiff polymers—such as DNA—and deciding on the type of position to target for a job search. The research outlined in the Genetic Code symposium summarized the current status of measuring and characterizing chromosomes. Even for chromosomes as simple as that of a bacteriophage, this presented a challenge in 1969. Chromosomes seemed clearly to be units containing the triplet genetic code organized in genes and aligned in a linear array. They were seen to vary enormously in length but whether each chromosome consisted of a continuous segment of DNA was unknown at the time. -
Allosteric Regulation of U1 Snrnp by Splicing Regulatory Proteins Controls
Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Allosteric Regulation of U1 snRNP by Splicing Regulatory Proteins Controls Spliceosomal Assembly. Hossein Shenasa1, Maliheh Movassat1, Elmira Forouzmand1 and Klemens J. Hertel1 1. Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, California Keywords: Spliceosomal Assembly, U1 snRNP, Splice Site Selection, Splicing Regulatory Proteins, Allosteric Regulation 1 Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Abstract: Alternative splicing is responsible for much of the transcriptomic and proteomic diversity observed in eukaryotes and involves combinatorial regulation by many cis-acting elements and trans-acting factors. SR and hnRNP splicing regulatory proteins often have opposing effects on splicing efficiency depending on where they bind the pre-mRNA relative to the splice site. Position-dependent splicing repression occurs at spliceosomal E-complex, suggesting that U1 snRNP binds but cannot facilitate higher order spliceosomal assembly. To test the hypothesis that the structure of U1 snRNA changes during activation or repression, we developed a method to structure-probe native U1 snRNP in enriched conformations that mimic activated or repressed spliceosomal E- complexes. While the core of U1 snRNA is highly structured, the 5' end of U1 snRNA shows different SHAPE reactivities and psoralen crosslinking efficiencies depending on where splicing regulatory elements are located relative to the 5' splice site. A motif within the 5' splice site binding region of U1 snRNA is more reactive towards SHAPE electrophiles when repressors are bound, suggesting U1 snRNA is bound, but less base paired. -
The RNA Splicing Response to DNA Damage
Biomolecules 2015, 5, 2935-2977; doi:10.3390/biom5042935 OPEN ACCESS biomolecules ISSN 2218-273X www.mdpi.com/journal/biomolecules/ Review The RNA Splicing Response to DNA Damage Lulzim Shkreta and Benoit Chabot * Département de Microbiologie et d’Infectiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC J1E 4K8, Canada; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-819-821-8000 (ext. 75321); Fax: +1-819-820-6831. Academic Editors: Wolf-Dietrich Heyer, Thomas Helleday and Fumio Hanaoka Received: 12 August 2015 / Accepted: 16 October 2015 / Published: 29 October 2015 Abstract: The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. -
Dramatically Reduced Spliceosome in Cyanidioschyzon Merolae
Dramatically reduced spliceosome in PNAS PLUS Cyanidioschyzon merolae Martha R. Starka, Elizabeth A. Dunnb, William S. C. Dunna, Cameron J. Grisdalec, Anthony R. Danielea, Matthew R. G. Halsteada, Naomi M. Fastc, and Stephen D. Radera,b,1 aDepartment of Chemistry, University of Northern British Columbia, Prince George, BC, V2N 4Z9 Canada; and Departments of bBiochemistry and Molecular Biology, and cBotany, University of British Columbia, Vancouver, BC, V6T 1Z4 Canada Edited by Joan A. Steitz, Howard Hughes Medical Institute, Yale University, New Haven, CT, and approved February 9, 2015 (received for review September 1, 2014) The human spliceosome is a large ribonucleoprotein complex that C. merolae is an acidophilic, unicellular red alga that grows at catalyzes pre-mRNA splicing. It consists of five snRNAs and more temperatures of up to 56 °C (6). At 16.5 million base pairs, its than 200 proteins. Because of this complexity, much work has genome is similar in size to that of S. cerevisiae and contains focusedontheSaccharomyces cerevisiae spliceosome, viewed as a comparable number of genes; however only one tenth as many a highly simplified system with fewer than half as many splicing introns were annotated in C. merolae: 26 intron-containing factors as humans. Nevertheless, it has been difficult to ascribe genes, 0.5% of the genome (6). The small number of introns in a mechanistic function to individual splicing factors or even to dis- C. merolae raises the questions of whether the full complexity cern which are critical for catalyzing the splicing reaction. We have of the canonical splicing machinery has been maintained or C merolae identified and characterized the splicing machinery from the red alga whether . -
Glossary of Terms
GLOSSARY OF TERMS Table of Contents A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | R | S | T | U | V | W | X | Y | Z A Amino acids: any of a class of 20 molecules that are combined to form proteins in living things. The sequence of amino acids in a protein and hence protein function are determined by the genetic code. From http://www.geneticalliance.org.uk/glossary.htm#C • The building blocks of proteins, there are 20 different amino acids. From https://www.yourgenome.org/glossary/amino-acid Antisense: Antisense nucleotides are strings of RNA or DNA that are complementary to "sense" strands of nucleotides. They bind to and inactivate these sense strands. They have been used in research, and may become useful for therapy of certain diseases (See Gene silencing). From http://www.encyclopedia.com/topic/Antisense_DNA.aspx. Antisense and RNA interference are referred as gene knockdown technologies: the transcription of the gene is unaffected; however, gene expression, i.e. protein synthesis (translation), is lost because messenger RNA molecules become unstable or inaccessible. Furthermore, RNA interference is based on naturally occurring phenomenon known as Post-Transcriptional Gene Silencing. From http://www.ncbi.nlm.nih.gov/probe/docs/applsilencing/ B Biobank: A biobank is a large, organised collection of samples, usually human, used for research. Biobanks catalogue and store samples using genetic, clinical, and other characteristics such as age, gender, blood type, and ethnicity. Some samples are also categorised according to environmental factors, such as whether the donor had been exposed to some substance that can affect health. -
Structural Insights Into Nuclear Pre-Mrna Splicing in Higher Eukaryotes
Downloaded from http://cshperspectives.cshlp.org/ on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Structural Insights into Nuclear pre-mRNA Splicing in Higher Eukaryotes Berthold Kastner,1 Cindy L. Will,1 Holger Stark,2 and Reinhard Lührmann1 1Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany 2Department of Structural Dynamics, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany Correspondence: [email protected] SUMMARY The spliceosome is a highly complex, dynamic ribonucleoprotein molecular machine that undergoes numerous structural and compositional rearrangements that lead to the formation of its active site. Recent advances in cyroelectron microscopy (cryo-EM) have provided a plethora of near-atomic structural information about the inner workings of the spliceosome. Aided by previous biochemical, structural, and functional studies, cryo-EM has confirmed or provided a structural basis for most of the prevailing models of spliceosome function, but at the same time allowed novel insights into splicing catalysis and the intriguing dynamics of the spliceosome. The mechanism of pre-mRNA splicing is highly conserved between humans and yeast, but the compositional dynamics and ribonucleoprotein (RNP) remodeling of the human spliceosome are more complex. Here, we summarize recent advances in our understanding of the molec- ular architecture of the human spliceosome, highlighting differences between the human and yeast -
Regulation of Pre-Mrna Splicing and Mrna Degradation in Saccharomyces Cerevisiae
Regulation of pre-mRNA splicing and mRNA degradation in Saccharomyces cerevisiae Yang Zhou Department of Molecular Biology This work is protected by the Swedish Copyright Legislation (Act 1960:729) Dissertation for PhD ISBN: 978-91-7601-749-4 Cover photo by Yang Zhou Electronic version available at: http://umu.diva-portal.org/ Printed by: Print & Media Umeå Umeå, Sweden 2017 by 千利休 Every single encounter never repeats in a life time. -Sen no Rikyu Table of Contents ABSTRACT ......................................................................................... ii APPENDED PAPERS .......................................................................... iii INTRODUCTION ................................................................................... 1 Pre-mRNA splicing ........................................................................................................... 1 Splicing and introns .................................................................................................... 1 The pre-mRNA Retention and splicing complex ...................................................... 6 Nuclear export of mRNAs................................................................................................. 7 Translation ........................................................................................................................ 7 Translation initiation ................................................................................................... 9 General mRNA degradation .......................................................................................... -
Post-Transcriptional Modification of Spliceosomal Rnas Is Normal in SMN-Deficient Cells
Downloaded from rnajournal.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press REPORT Post-transcriptional modification of spliceosomal RNAs is normal in SMN-deficient cells SVETLANA DERYUSHEVA,1,3 MARIA CHOLEZA,2,3 ADRIEN BARBAROSSA,2 JOSEPH G. GALL,1 and RE´MY BORDONNE´2,4 1Carnegie Institution, Baltimore, Maryland 21218, USA 2Institut de Ge´ne´tique Mole´culaire de Montpellier (IGMM), CNRS UMR 5535/IFR122, Universite´ Montpellier, 34293 Montpellier Cedex 5, France ABSTRACT The survival of motor neuron (SMN) protein plays an important role in the biogenesis of spliceosomal snRNPs and is one factor required for the integrity of nuclear Cajal bodies (CBs). CBs are enriched in small CB-specific (sca) RNAs, which guide the formation of pseudouridylated and 29-O-methylated residues in the snRNAs. Because SMN-deficient cells lack typical CBs, we asked whether the modification of internal residues of major and minor snRNAs is defective in these cells. We mapped modified nucleotides in the major U2 and the minor U4atac and U12 snRNAs. Using both radioactive and fluorescent primer extension approaches, we found that modification of major and minor spliceosomal snRNAs is normal in SMN-deficient cells. Our experiments also revealed a previously undetected pseudouridine at position 60 in human U2 and 29-O-methylation of A1, A2, and G19 in human U4atac. These results confirm, and extend to minor snRNAs, previous experiments showing that scaRNPs can function in the absence of typical CBs. Furthermore, they show that the differential splicing defects in SMN-deficient cells are not due to failure of post-transcriptional modification of either major or minor snRNAs.