DOCSLIB.ORG

An Exonic Splicing Silencer Is Involved in the Regulated Splicing of Glucose 6-Phosphate Dehydrogenase Mrna Wioletta Szeszel-Fedorowicz

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
An Exonic Splicing Silencer Is Involved in the Regulated Splicing of Glucose 6-Phosphate Dehydrogenase Mrna Wioletta Szeszel-Fedorowicz Faculty Scholarship 2006 An Exonic Splicing Silencer Is Involved In The Regulated Splicing Of Glucose 6-Phosphate Dehydrogenase Mrna Wioletta Szeszel-Fedorowicz Indrani Talukdar Brian N. Griffith Callee M. Walsh Lisa M. Salati Follow this and additional works at: https://researchrepository.wvu.edu/faculty_publications Digital Commons Citation Szeszel-Fedorowicz, Wioletta; Talukdar, Indrani; Griffith,r B ian N.; Walsh, Callee M.; and Salati, Lisa M., "An Exonic Splicing Silencer Is Involved In The Regulated Splicing Of Glucose 6-Phosphate Dehydrogenase Mrna" (2006). Faculty Scholarship. 464. https://researchrepository.wvu.edu/faculty_publications/464 This Article is brought to you for free and open access by The Research Repository @ WVU. It has been accepted for inclusion in Faculty Scholarship by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 45, pp. 34146–34158, November 10, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. An Exonic Splicing Silencer Is Involved in the Regulated Splicing of Glucose 6-Phosphate Dehydrogenase mRNA* Received for publication, April 20, 2006, and in revised form, September 14, 2006 Published, JBC Papers in Press, September 15, 2006, DOI 10.1074/jbc.M603825200 Wioletta Szeszel-Fedorowicz, Indrani Talukdar, Brian N. Griffith, Callee M. Walsh, and Lisa M. Salati1 From the Department of Biochemistry and Molecular Pharmacology, West Virginia University, Morgantown, West Virginia 26506 The inhibition of glucose-6-phosphate dehydrogenase (G6PD) in converting excess dietary energy into a storage form. Con- expression by arachidonic acid occurs by changes in the rate sistent with this role in energy homeostasis, the capacity of this of pre-mRNA splicing. Here, we have identified a cis-acting pathway is regulated by dietary changes, such as fasting, feed- RNA element required for regulated splicing of G6PD mRNA. ing, and the amount and type of carbohydrate and polyunsatu- Using transfection of G6PD RNA reporter constructs into rat rated fat in the diet (1). For many of the lipogenic enzymes, hepatocytes, the cis-acting RNA element involved in this regu- regulation of enzyme amount occurs primarily by changes in lation was localized to nucleotides 43–72 of exon 12 in the G6PD the transcription rate of the gene, but posttranscriptional reg- mRNA. In in vitro splicing assays, RNA substrates containing ulation via mRNA stability has also been implicated (1). G6PD exon 12 were not spliced. In contrast, RNA substrates contain- differs from the other family members in that dietary regulation ing other regions (exons 8 and 9 or exons 10 and 11) of the G6PD occurs exclusively at a posttranscriptional step (2–4). mRNA were efficiently spliced. Furthermore, exon 12 can G6PD expression is inhibited by polyunsaturated fatty inhibit splicing when substituted for other exons in RNA sub- acids, such as arachidonic acid; this occurs at a unique post- strates that are readily spliced. This activity of the exon 12 reg- transcriptional step involving a decrease in the rate of splic- ulatory element suggests that it is an exonic splicing silencer. ing of the nascent G6PD transcript. Several lines of evidence Consistent with its activity as a splicing silencer, spliceosome indicate that changes in mature mRNA accumulation are assembly was inhibited on RNA substrates containing exon 12 caused by changes in the efficiency of splicing of the G6PD compared with RNAs representing other regions of the G6PD transcript. First, changes in the cytoplasmic accumulation of transcript. Elimination of nucleotides 43–72 of exon 12 did not G6PD mRNA are preceded by changes in the accumulation restore splicing of exon 12-containing RNA; thus, the 30-nucle- of mRNA in the nucleus in the absence of changes in tran- otide element may not be exclusively a silencer. The binding of scriptional activity of the gene (3–5). Second, stimulatory heterogeneous nuclear ribonucleoproteins K, L, and A2/B1 treatments, such as refeeding, enhance the rate of accumu- from both HeLa and hepatocyte nuclear extracts to the element lation of partially and fully spliced G6PD mRNA with little or further supports its activity as a silencer. In addition, SR pro- no change in abundance of the primary transcript (2). Third, teins bind to the element, consistent with the presence of inhibition of G6PD expression by polyunsaturated fat results enhancer activity within this sequence. Thus, an exonic splicing in an accumulation of partially spliced mRNA in the nucleus silencer is involved in the inhibition of splicing of a constitu- despite a decrease in the nuclear content of mature mRNA tively spliced exon in the G6PD mRNA. (6). Finally, using transient transfection of RNA reporter constructs containing progressive deletion of the 13 exons of the G6PD precursor mRNA (pre-mRNA) into rat hepato- Glucose-6-phosphate dehydrogenase (G6PD)2 is a member cytes, we have demonstrated that exon 12 and its surround- of a family of enzymes that catalyze the de novo synthesis of ing introns are necessary for the arachidonic acid inhibition fatty acids. In liver, the lipogenic pathway plays an essential role of mRNA accumulation (6). Thus, we conclude that G6PD expression is regulated by changes in the rate of pre-mRNA * This work was supported by National Institutes of Health Grant DK46897 (to splicing. L. M. S.), American Heart Association Grant 0315129B (to B. N. G.), the This mechanism of regulation of G6PD by inhibition of its COBRE for Signal Transduction and Cancer (National Institutes of Health splicing may be shared across other metabolic genes, even those Grant P20RR016440), and the West Virginia University Proteomics Facility. The costs of publication of this article were defrayed in part by the pay- regulated by transcriptional mechanisms. In this regard, nutri- ment of page charges. This article must therefore be hereby marked tional status regulates expression of the tricarboxylate (citrate) “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indi- carrier and spot 14, a putative lipogenic protein by a nuclear cate this fact. 1 To whom correspondence should be addressed: Dept. of Biochemistry and posttranscriptional mechanism (2, 7–9). Regulation of accu- Molecular Pharmacology, West Virginia University Health Sciences Center, mulation of spliced intermediates has been observed for these P.O. Box 9142, Morgantown, WV 26506. Tel.: 304-293-7759; E-mail: Lsalati@ genes, but the molecular details are not known. Regulating gene hsc.wvu.edu. expression by changes in the rate of pre-mRNA splicing in addi- 2 The abbreviations used are: G6PD, glucose-6-phosphate dehydrogen- ase; pre-mRNA, precursor-mRNA; ESE, exonic splicing enhancer; SR, ser- tion to transcriptional regulation would result in a more rapid ine/arginine-rich; snRNP, small nuclear ribonucleoprotein particle; response to nutritional or hormonal changes. hnRNP, heterogeneous nuclear ribonucleoprotein; ESS, exonic splicing Pre-mRNA splicing results in the removal of introns from silencer; CMV, cytomegalovirus; UTR, untranslated region; U2AF, U2 primary transcripts and the joining of the exons containing auxiliary factor; Xcorr, cross-correlation; nt, nucleotide; LC, liquid chro- matography; MS, mass spectrometry. the protein coding information. The G6PD gene is over 18 34146 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281•NUMBER 45•NOVEMBER 10, 2006 An Exonic Splicing Silencer in a Constitutively Spliced Exon kb, and all of its exons are constitutively spliced. Exons can attenuating the formation of the first ATP-requiring complex be constitutively or alternatively spliced. Constitutively in this process and binds hnRNPs, associated with inhibition of spliced exons are those always present in the mRNA and splicing. This is the first report of an ESS in a constitutively account for the majority of exons. The alternative splicing of spliced exon being involved in the regulation of gene mRNA transcripts, which occurs for at least 60% of all genes, expression. results in the production of different proteins and can occur in a tissue- or treatment-specific manner (10). Both consti- EXPERIMENTAL PROCEDURES tutive and alternative splicing requires distinguishing exons Animal Care and Cell Culture—Male Sprague-Dawley rats from the surrounding intronic RNA. Exon definition, as the (175–250 g) were fed a chow diet and then starved 16 h prior to process of exon recognition is called, utilizes intronic hepatocyte isolation. Hepatocytes were isolated by a modifica- sequences, which include the 5Ј and 3Ј splice sites, polypy- tion of the Seglen technique (25) as described previously (4). rimidine tract, and branch point, as well as sequence ele- Hepatocytes (3.1 ϫ 106) were placed in 60-mm dishes coated ments in the exon called exonic splicing enhancers (ESEs) with rat tail collagen and incubated in Hi/Wo/BA medium (11). ESEs are not highly conserved with respect to sequence (Waymouth MB752/l plus 20 mM Hepes, pH 7.4, 0.5 mM serine, and thus must be functionally characterized in the 0.5 mM alanine, 0.2% bovine serum albumin) plus 5% newborn pre-mRNA (12, 13). The activity of ESEs involves their bind- calf serum (37 °C, 5% CO2). Cell viability in all experiments was ing by members of a family of splicing regulatory proteins, 90% or greater as estimated by trypan blue (0.04%) exclusion. serine/arginine-rich (SR) proteins (14). SR proteins partici- After 3–4 h, the medium was replaced with serum-free pate in splicing by enhancing the recruitment of the key medium, and the constructs presented in Figs. 1 and 2 were spliceosome components, U1 small nuclear ribonucleopro- transfected. Each construct (2.5 ␮g/plate) was transfected using tein particle (snRNP) and U2 snRNP (11). In the absence of Lipofectin according to the manufacturer’s protocol (Invitro- an ESE, binding of U1 and U2 snRNPs to the 5Ј splice site and gen). The ratio of DNA to liposome reagent was 1:6.7.
Load more

Recommended publications
  • In Silico Tools for Splicing Defect Prediction: a Survey from the Viewpoint of End Users
    © American College of Medical Genetics and Genomics REVIEW In silico tools for splicing defect prediction: a survey from the viewpoint of end users Xueqiu Jian, MPH1, Eric Boerwinkle, PhD1,2 and Xiaoming Liu, PhD1 RNA splicing is the process during which introns are excised and informaticians in relevant areas who are working on huge data sets exons are spliced. The precise recognition of splicing signals is critical may also benefit from this review. Specifically, we focus on those tools to this process, and mutations affecting splicing comprise a consider- whose primary goal is to predict the impact of mutations within the able proportion of genetic disease etiology. Analysis of RNA samples 5′ and 3′ splicing consensus regions: the algorithms used by different from the patient is the most straightforward and reliable method to tools as well as their major advantages and disadvantages are briefly detect splicing defects. However, currently, the technical limitation introduced; the formats of their input and output are summarized; prohibits its use in routine clinical practice. In silico tools that predict and the interpretation, evaluation, and prospection are also discussed. potential consequences of splicing mutations may be useful in daily Genet Med advance online publication 21 November 2013 diagnostic activities. In this review, we provide medical geneticists with some basic insights into some of the most popular in silico tools Key Words: bioinformatics; end user; in silico prediction tool; for splicing defect prediction, from the viewpoint of end users. Bio- medical genetics; splicing consensus region; splicing mutation INTRODUCTION TO PRE-mRNA SPLICING AND small nuclear ribonucleoproteins and more than 150 proteins, MUTATIONS AFFECTING SPLICING serine/arginine-rich (SR) proteins, heterogeneous nuclear ribo- Sixty years ago, the milestone discovery of the double-helix nucleoproteins, and the regulatory complex (Figure 1).
    [Show full text]
  • Position-Dependent Splicing Activation and Repression by SR and Hnrnp Proteins Rely on Common Mechanisms
    Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanisms STEFFEN ERKELENZ,1,3 WILLIAM F. MUELLER,2,3 MELANIE S. EVANS,2 ANKE BUSCH,2 KATRIN SCHO¨ NEWEIS,1 KLEMENS J. HERTEL,2,4 and HEINER SCHAAL1,4 1Institute of Virology, Heinrich-Heine-University, D-40225 Du¨sseldorf, Germany 2Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, California 92697-4025, USA ABSTRACT Alternative splicing is regulated by splicing factors that modulate splice site selection. In some cases, however, splicing factors show antagonistic activities by either activating or repressing splicing. Here, we show that these opposing outcomes are based on their binding location relative to regulated 59 splice sites. SR proteins enhance splicing only when they are recruited to the exon. However, they interfere with splicing by simply relocating them to the opposite intronic side of the splice site. hnRNP splicing factors display analogous opposing activities, but in a reversed position dependence. Activation by SR or hnRNP proteins increases splice site recognition at the earliest steps of exon definition, whereas splicing repression promotes the assembly of nonproductive complexes that arrest spliceosome assembly prior to splice site pairing. Thus, SR and hnRNP splicing factors exploit similar mechanisms to positively or negatively influence splice site selection. Keywords: SR protein; hnRNP protein; pre-mRNA splicing; splicing activation; splicing repression INTRODUCTION their RS domain (Fu and Maniatis 1992; Lin and Fu 2007) or through RNA binding domain interactions with U1 Alternative pre-mRNA splicing promotes high proteomic snRNP-specific protein 70K (Cho et al. 2011). However, in diversity despite the limited number of protein coding some cases SR proteins were shown to interfere with exon genes (Nilsen and Graveley 2010).
    [Show full text]
  • Determinants of Exon 7 Splicing in the Spinal Muscular Atrophy Genes, SMN1 and SMN2 Luca Cartegni,*,† Michelle L
    Determinants of Exon 7 Splicing in the Spinal Muscular Atrophy Genes, SMN1 and SMN2 Luca Cartegni,*,† Michelle L. Hastings,* John A. Calarco,‡ Elisa de Stanchina, and Adrian R. Krainer Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Spinal muscular atrophy is a neurodegenerative disorder caused by the deletion or mutation of the survival-of- motor-neuron gene, SMN1. An SMN1 paralog, SMN2, differs by a CrT transition in exon 7 that causes substantial skipping of this exon, such that SMN2 expresses only low levels of functional protein. A better understanding of SMN splicing mechanisms should facilitate the development of drugs that increase survival motor neuron (SMN) protein levels by improving SMN2 exon 7 inclusion. In addition, exonic mutations that cause defective splicing give rise to many genetic diseases, and the SMN1/2 system is a useful paradigm for understanding exon-identity determinants and alternative-splicing mechanisms. Skipping of SMN2 exon 7 was previously attributed either to the loss of an SF2/ASF–dependent exonic splicing enhancer or to the creation of an hnRNP A/B–dependent exonic splicing silencer, as a result of the CrT transition. We report the extensive testing of the enhancer-loss and silencer- gain models by mutagenesis, RNA interference, overexpression, RNA splicing, and RNA-protein interaction ex- periments. Our results support the enhancer-loss model but also demonstrate that hnRNP A/B proteins antagonize SF2/ASF–dependent ESE activity and promote exon 7 skipping by a mechanism that is independent of the CrT transition and is, therefore, common to both SMN1 and SMN2. Our findings explain the basis of defective SMN2 splicing, illustrate the fine balance between positive and negative determinants of exon identity and alternative splicing, and underscore the importance of antagonistic splicing factors and exonic elements in a disease context.
    [Show full text]
  • HIV-1: to Splice Or Not to Splice, That Is the Question
    viruses Review HIV-1: To Splice or Not to Splice, That Is the Question Ann Emery 1 and Ronald Swanstrom 1,2,3,* 1 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA; [email protected] 2 Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA 3 Center for AIDS Research, University of North Carolina, Chapel Hill, NC 27599, USA * Correspondence: [email protected] Abstract: The transcription of the HIV-1 provirus results in only one type of transcript—full length genomic RNA. To make the mRNA transcripts for the accessory proteins Tat and Rev, the genomic RNA must completely splice. The mRNA transcripts for Vif, Vpr, and Env must undergo splicing but not completely. Genomic RNA (which also functions as mRNA for the Gag and Gag/Pro/Pol precursor polyproteins) must not splice at all. HIV-1 can tolerate a surprising range in the relative abundance of individual transcript types, and a surprising amount of aberrant and even odd splicing; however, it must not over-splice, which results in the loss of full-length genomic RNA and has a dramatic fitness cost. Cells typically do not tolerate unspliced/incompletely spliced transcripts, so HIV-1 must circumvent this cell policing mechanism to allow some splicing while suppressing most. Splicing is controlled by RNA secondary structure, cis-acting regulatory sequences which bind splicing factors, and the viral protein Rev. There is still much work to be done to clarify the combinatorial effects of these splicing regulators. These control mechanisms represent attractive targets to induce over-splicing as an antiviral strategy.
    [Show full text]
  • Free PDF Download
    Eur opean Rev iew for Med ical and Pharmacol ogical Sci ences 2014; 18: 526-536 Bioinformatics analysis of Exonic Splicing Enhancers (ESEs) for predicting potential regulatory elements of hTERT mRNA Splicing F. WANG 1, G.-M. CHANG 2, X. GENG 3,4,5 1Department of Neurology, General Hospital, Tianjin Medical University, China 2Department of Clinical Laboratory, General Hospital, Tianjin Medical University, China 3Department of Biochemistry and Molecular Biology, Tianjin Medical University, China 4Tianjin Key Laboratory of Cellular and Molecular Immunology, China 5Key Laboratory of Educational Ministry of China Abstract. – OBJECTIVES : Alternative splicing Introduction of human telomerase reverse transcriptase (hTERT) has an important effect on regulating Telomeres are specialized structures located at telomerase activity. Exonic splicing enhancers (ESEs) are a family of conserved splicing factors the ends of eukaryotic chromosomes, that pro - that participate in multiple steps of the splicing tect linear chromosome ends from unwanted re - pathway. Our aim is to analyze the ESEs for pre - pair or recombination . Progressive telomere dicting the potential regulatory elements of shortening occurs in somatic cells due to incom - hTERT mRNA splicing. plete replication of chromosome ends. Telom - MATERIALS AND METHODS: Enter the FAS - erase, a ribonucleoprotein complex, is capable of TA format of hTERT total sequences or individ - adding telomere repeats to the 3 ’ end, which is ual exon as the input data in the main interface essential for the telomere length maintenance in of ESEfinder3.0 and ESEfinder2.0 program. Ana - 1 lyze the data of output results and compare the germ cells and stem cells . Human telomerase differences between ESEfinder3.0 and ESEfind - holoenzyme consists of three components: a er2.0 program.
    [Show full text]
  • Systematic Identification and Analysis of Exonic Splicing Silencers
    Cell, Vol. 119, 831–845, December 17, 2004, Copyright ©2004 by Cell Press Systematic Identification and Analysis of Exonic Splicing Silencers Zefeng Wang,1 Michael E. Rolish,1,2 these imposters implies that sequence features outside Gene Yeo,1,3 Vivian Tung,1 of the canonical splice site/branch site elements must Matthew Mawson,1 and Christopher B. Burge1,* play important roles in splicing of most or all transcripts. 1Department of Biology Prime candidates for these features are exonic or in- 2 Department of Electrical Engineering tronic cis-elements that either enhance or silence the and Computer Science usage of adjacent splice sites. 3 Department of Brain and Cognitive Sciences Two major classes of cis-regulators of splicing are the Massachusetts Institute of Technology exonic splicing enhancers (ESEs) and exonic splicing Cambridge, Massachusetts 02139 silencers (ESSs). These elements generally function by recruiting protein factors that interact favorably or unfa- vorably with components of the core splicing machinery Summary such as U1 and U2 snRNPs (Kohtz et al., 1994; Wu and Maniatis, 1993). Several groups of ESEs are known, Exonic splicing silencers (ESSs) are cis-regulatory including purine-rich and AC-rich elements, as well as elements that inhibit the use of adjacent splice sites, some with more complex composition (Graveley, 2000; often contributing to alternative splicing (AS). To sys- Zheng, 2004). Most known ESEs function by recruiting tematically identify ESSs, an in vivo splicing reporter members of the serine-arginine-rich (SR) protein family, system was developed to screen a library of random which interact favorably with each other, with the pre- decanucleotides.
    [Show full text]
  • Progress in Therapeutic Antisense Applications for Neuromuscular Disorders
    European Journal of Human Genetics (2010) 18, 146–153 & 2010 Macmillan Publishers Limited All rights reserved 1018-4813/10 $32.00 www.nature.com/ejhg REVIEW Progress in therapeutic antisense applications for neuromuscular disorders Annemieke Aartsma-Rus*,1 and Gert-Jan B van Ommen1 Neuromuscular disorders are a frequent cause of chronic disability in man. They often result from mutations in single genes and are thus, in principle, well suited for gene therapy. However, the tissues involved (muscle and the central nervous system) are post-mitotic, which poses a challenge for most viral vectors. In some cases, alternative approaches may use small molecules, for example, antisense oligonucleotides (AONs). These do not deliver a new gene, but rather modulate existing gene products or alter the utilization of pathways. For Duchenne muscular dystrophy, this approach is in early phase clinical trials, and for two other common neuromuscular disorders (spinal muscular atrophy and myotonic dystrophy), significant preclinical advances have recently been made. European Journal of Human Genetics (2010) 18, 146–153; doi:10.1038/ejhg.2009.160; published online 7 October 2009 Keywords: Duchenne muscular dystrophy; spinal muscular atrophy; myotonic dystrophy; therapy; antisense oligonucleotide; exon skipping INTRODUCTION translational trajectories.5–8 Interestingly, although the tool is the same Neuromuscular disorders (NMDs) are a frequent cause of loss of for each disease (AONs), it is tailored in different ways for the different ambulation, chronic
    [Show full text]
  • Using Positional Distribution to Identify Splicing Elements and Predict Pre-Mrna Processing Defects in Human Genes
    Using positional distribution to identify splicing elements and predict pre-mRNA processing defects in human genes Kian Huat Lima, Luciana Ferrarisa, Madeleine E. Fillouxa, Benjamin J. Raphaelb,c, and William G. Fairbrothera,c,d,1 aDepartment of Molecular and Cellular Biology and Biochemistry, Brown University, 70 Ship Street, Providence, RI 02903; bDepartment of Computer Science, Brown University, Providence, RI 02912; cCenter for Computational Molecular Biology, 151 Waterman Street, Providence, RI 02912; and dCenter for Genomics and Proteomics, Brown University, 70 Ship Street, Providence, RI 02903 Edited by Phillip A. Sharp, MIT, Cambridge, MA, and approved May 13, 2011 (received for review February 11, 2011) We present an intuitive strategy for predicting the effect of molecular intervention. As physical methods for the detection of sequence variation on splicing. In contrast to transcriptional ele- alternative splicing require large panels of genotyped accessible ments, splicing elements appear to be strongly position dependent. tissue, these studies will probably continue to be limited to sam- We demonstrated that exonic binding of the normally intronic spli- ples harvested from human blood. An alternate approach is the cing factor, U2AF65, inhibits splicing. Reasoning that the positional prediction of causative variations from single-nucleo polymorph- distribution of a splicing element is a signature of its function, we ism (SNPs) that fall within splicing elements. The key to this developed a method for organizing all possible sequence motifs approach is being able to identify what the splicing elements into clusters based on the genomic profile of their positional dis- are and whether a variation is disruptive. tribution around splice sites.
    [Show full text]
  • Alternative Splicing
    Alternative Splicing Multiple control Mechanisms & Involvement in human disease Anabella Srebrow Laboratorio de Fisiología y Biología Molecular IFIBYNE-CONICET Facultad de Ciencias Exactas y Naturales Universidad de Buenos Aires outside cytoplasm nucleus protein-coding genes Cleavage and Polyadenylation Capping Splicing Co-transcriptional processing post-transcriptional co-transcriptional pre-mRNA processing pre-mRNA processing Pol II Pol II Transcription Capping Transcription Splicing Polyadenylation Primary transcript CAP Capping Splicing Polyadenylation An CAP An CAP Co-transcriptional processing • requires coordination between molecular machineries acting at different steps • coordination is mainly achieved by the function of RNA polymerase II CTD (Carboxi Terminal Domain) multiple repeats of “YSPTSPS” heptad Serine phosphorylations are critical for transcription & association with processing factors mRNA factory governed by pol II CTD capping polyadenylation factors factors P P P P P P P Splicing factors CTD (YSPTSPS)52 Bentley lab: McCracken et al., Nature 1997 Figure 6-23 Molecular Biology of the Cell (© Garland Science 2008) Pol II CTD is crucial for pre-mRNA processing TRANSCRIPTION CAPPING SPLICING polyA Pol III Normal Inhibited Inhibited Inhibited Cleveland lab: Sisodia et al., MCB 1987 Normal Normal Normal Normal Normal* Inhibited Inhibited Inhibited Bentley lab: McCracken et al., Nature 1997 What is Splicing ? AUG STOP Lynch, Nature Reviews Immunol. 2004 Sequences required for splicing 30-50 nt exon exon a c c a c ...AGgu agu........................//.... x u a ...//…………..ccucuucucuucuccuxcagG..... g u u g u Branch site Polypyrimidine ’ tract 5 splice site ’ (5’ss) 3 splice site (3’ss) DONOR SITE ACCEPTOR SITE The Spliceosome snRNP snRNP snRNP U5 U4 U6 snRNP U1 snRNP U2AF65 U2AF35 U2 BBP 30-50 nt exon exon a c c a c ...AGgu agu........................//...
    [Show full text]
  • Perspective in Alternative Splicing Coupled to Nonsense-Mediated Mrna Decay
    International Journal of Molecular Sciences Review Perspective in Alternative Splicing Coupled to Nonsense-Mediated mRNA Decay Juan F. García-Moreno 1,2 and Luísa Romão 1,2,* 1 Department of Human Genetics, Instituto Nacional de Saúde Doutor Ricardo Jorge, 1649-016 Lisboa, Portugal; [email protected] 2 Faculty of Science, BioISI—Biosystems and Integrative Sciences Institute, University of Lisboa, 1749-016 Lisboa, Portugal * Correspondence: [email protected]; Tel.: +351-217-508-155 Received: 29 September 2020; Accepted: 7 December 2020; Published: 10 December 2020 Abstract: Alternative splicing (AS) of precursor mRNA (pre-mRNA) is a cellular post-transcriptional process that generates protein isoform diversity. Nonsense-mediated RNA decay (NMD) is an mRNA surveillance pathway that recognizes and selectively degrades transcripts containing premature translation-termination codons (PTCs), thereby preventing the production of truncated proteins. Nevertheless, NMD also fine-tunes the gene expression of physiological mRNAs encoding full-length proteins. Interestingly, around one third of all AS events results in PTC-containing transcripts that undergo NMD. Numerous studies have reported a coordinated action between AS and NMD, in order to regulate the expression of several genes, especially those coding for RNA-binding proteins (RBPs). This coupling of AS to NMD (AS-NMD) is considered a gene expression tool that controls the ratio of productive to unproductive mRNA isoforms, ultimately degrading PTC-containing non-functional mRNAs. In this review, we focus on the mechanisms underlying AS-NMD, and how this regulatory process is able to control the homeostatic expression of numerous RBPs, including splicing factors, through auto- and cross-regulatory feedback loops.
    [Show full text]
  • Finding Signals That Regulate Alternative Splicing in the Post- Comment Genomic Era Andrea N Ladd and Thomas a Cooper
    http://genomebiology.com/2002/3/11/reviews/0008.1 Review Finding signals that regulate alternative splicing in the post- comment genomic era Andrea N Ladd and Thomas A Cooper Address: Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA. Correspondence: Thomas A Cooper. E-mail: [email protected] reviews Published: 23 October 2002 Genome Biology 2002, 3(11):reviews0008.1–0008.16 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2002/3/11/reviews/0008 © BioMed Central Ltd (Print ISSN 1465-6906; Online ISSN 1465-6914) reports Abstract Alternative splicing of pre-mRNAs is central to the generation of diversity from the relatively small number of genes in metazoan genomes. Auxiliary cis elements and trans-acting factors are required for the recognition of constitutive and alternatively spliced exons and their inclusion in pre-mRNA. Here, deposited research we discuss the regulatory elements that direct alternative splicing and how genome-wide analyses can aid in their identification. Alternative splicing, the process by which multiple mRNA (ESTs; sequenced portions of cDNAs) range from 30 to 60% isoforms are generated from a single pre-mRNA species, is of genes, and even 60% might be an underestimate [3] an important means of regulating gene expression. Alterna- because there is a lack of ESTs for many tissues and many refereed research tive splicing determines cell fate in numerous contexts, such developmental stages, and ESTs are biased toward the as sexual differentiation in Drosophila and apoptosis in 3؅ end of an mRNA.
    [Show full text]
  • Snapshot: Formation of Mrnps Megan Bergkessel, Gwendolyn M
    SnapShot: Formation of mRNPs Megan Bergkessel, Gwendolyn M. Wilmes, and Christine Guthrie Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA Capping • 7-methyl-guanosine cap added to 5′ end of RNA by 3 enzymes: RNA 5′ triphosphatase (RT), guanyltransferase (GT) (bifunctional RT-GT in vertebrates), and methyltransferase (MT). • Capping enzymes (RT and MT in yeast, RT/GT in vertebrates) recruited to RNA by Ser5-phosphorylated RNA Pol II CTD. • Cap added when ~20 nt of RNA is transcribed. Cap addition coupled to promoter clearance and early transcription elongation. • Nuclear cap-binding proteins (Cbc2-Sto1 and Cbp80-20) replaced by cytoplasmic CBPs (Cdc33/eIF4E) after export. Splicing • Pre-mRNA splicing, a two-step trans-esterifi cation reaction with a branched intermediate, is mediated by 5 snRNAs (in snRNPs) and 80–200 proteins. U1 snRNP, BBP/ SF1, and Mud2/U2AF recognize RNA elements to form a commitment complex. U2 snRNP joins next, followed by U5, U4/U6 snRNPs. After major rearrangements, catalytically active spliceosome contains U2, U5, and U6. • 8 DExH/D-box helicases (Prp5/DDX23, Sub2/UAP56, Prp28/DDX46, Brr2/U5-200KD, Prp2/DHX16, Prp16/ DHX38, Prp22/DHX8, Prp43/DHX15) and a GTPase (Snu114/SNRP116) facilitate rearrangements and regulate RNA Elements reaction fi delity. 5′ss: /GURAG RNA Elements • Vertebrate exon-exon junctions marked by EJC, which BP: YURAY triggers NMD when downstream of a stop codon. 5′ss (5′ splice site): /GUAUGU 3′ss: AG/ • Splicing coupled to mRNP export via proteins that act in BP (branchpoint): UACUAAC preceded by long both processes, such as Sub2/UAP56, serine-arginine rich 3′ss (3′ splice site): AG/ Y tract preceded by short Y tract (SR) proteins, and hnRNPs.
    [Show full text]