DOCSLIB.ORG

Localization of an Exonic Splicing Enhancer Responsible For

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Localization of an Exonic Splicing Enhancer Responsible For 3108–3115 Nucleic Acids Research, 2001, Vol. 29, No. 14 © 2001 Oxford University Press Localization of an exonic splicing enhancer responsible for mammalian natural trans-splicing Concha Caudevilla, Carles Codony1, Dolors Serra, Guillem Plasencia, Ruth Román, Adolf Graessmann2, Guillermina Asins, Montserrat Bach-Elias1 and Fausto G. Hegardt* Department of Biochemistry and Molecular Biology, School of Pharmacy, Diagonal 643, University of Barcelona, 08028 Barcelona, Spain, 1IIBB Consejo Superior de Investigaciones Científicas, 08034 Barcelona, Spain and 2Institut für Molekularbiologie, Freie Universität Berlin, D-14195 Berlin, Germany Received February 15, 2001; Revised and Accepted June 1, 2001 DDBJ/EMBL/GenBank accession nos AF144397–AF144399 ABSTRACT recognize exon–intron boundaries at both 5′ and 3′ splice sites and then produce the necessary reactions to ligate the exons Carnitine octanoyltransferase (COT) produces three involved. Two kinds of splicing are observed in nature. different transcripts in rat through cis-andtrans- Cis-splicing, by which introns from the same pre-mRNA are splicing reactions, which may lead to the synthesis removed, and trans-splicing, in which two pre-mRNAs are of two proteins. Generation of the three COT tran- processed to produce a mature transcript that contains exons scripts in rat does not depend on sex, development, from both precursors. The trans-splicing process has been fat feeding, the inclusion of the peroxisome prolifer- described mostly in trypanosome, nematodes, plant/algal ator diethylhexyl phthalate in the diet or hyper- chloroplasts and plant mitochondria (1). insulinemia. In addition, trans-splicing was not A family of serine–arginine-rich RNA binding proteins, detected in COT of other mammals, such as human, known as SR proteins, plays a critical role in the formation of pig, cow and mouse, or in Cos7 cells from monkey. active spliceosomes. The SR proteins recognize exonic Rat COT exon 2 contains two purine-rich sequences. splicing enhancers (ESEs) promoting both constitutive and Mutation of the rat COT exon 2 upstream box does regulated splicing by bringing the flanking splice sites. Trans-splicing efficiency may correlate with the presence of notaffectthetrans-splicing in vitro between two these ESE sequences (2,3). Trans-splicing in vitro only occurs truncated constructs containing exon 2 and its when the 3′ exon contains ESE sequences (4). adjacent intron boundaries. In contrast, mutation of In the course of our research, natural trans-splicing was the downstream box from the rat sequence observed in rat carnitine octanoyltransferase (COT) pre-mRNAs (GAAGAAG) to a random sequence or the sequence (5). This mechanism produces three different transcripts: one observed in the other mammals (AAAAAAA) produced by cis-splicing and two produced by trans-splicing, decreased trans-splicing in vitro. In contrast, muta- in which exons 2 and 3 are repeated. Western blot analysis of tion of the AAAAAAA box of human COT exon 2 to peroxisomal proteins revealed two translated proteins with GAAGAAG increases trans-splicing. Heterologous molecular masses which may correspond to the cis-spliced as reactions between COT exon 2 from rat and human well as the trans-spliced RNAs in which exons 2 and 3 are do not produce trans-splicing. HeLa cells transfected repeated. Recently, trans-splicing corresponding to other with minigenes of rat COT sequences produced cis- genes has been observed in mammals. In some cases, mature mRNAs containing a mixture of exons from different genes and trans-spliced bands. Mutation of the GAAGAAG have been observed; this transcript heterogeneity may box to AAAAAAA abolished trans-splicing and contribute to phenotypic variation in mammals (6–12). decreased cis-splicing in vivo. We conclude that In this report we show that, in contrast to rat, other mammals GAAGAAG is an exonic splicing enhancer that could such as human, mouse, pig, cow and Cos7 cells of monkey do induce natural trans-splicing in rat COT. not produce detectable trans-splicing of the COT gene. We also show that trans-splicing in rat COT is not due to any metabolic, nutritional, hormonal or developmental condition. INTRODUCTION Trans-splicing in rat is attributable in part to the occurrence of The precise removal of introns from pre-mRNA by splicing is an ESE sequence (GAAGAAG). Trans-splicing is not found in an important step in the expression of genes. It requires the other mammals, which have a conserved sequence, interaction of several proteins and small ribonucleoprotein AAAAAAA, instead. Splicing experiments in vitro,inwhich particles, which all function in concert. These components the rat putative ESE sequence (GAAGAAG) was mutated *To whom correspondence should be addressed. Tel: +34 93 402 4523; Fax: +34 93 402 4520; Email: [email protected] The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors Nucleic Acids Research, 2001, Vol. 29, No. 14 3109 either to a random sequence or to AAAAAAA, revealed a was also prepared by PCR taking construct B as template and marked decrease in trans-splicing of the COT pre-mRNA. primers I1.f (5′-CATGGGGTGCTAAATCCACA-3′)and Moreover, when the human COT exon 2 box AAAAAAA was MutESE2.r (5′-CTGACTCAAGGTACAGATGCAGTGAT- mutated to GAAGAAG in vitro a marked increase in TCTTC-3′) and subcloned into Bluescript SK+ digested with trans-splicing was observed. In vivo experiments also showed EcoRV. The sequences underlined above indicate the mutated that mutation of the GAAGAAG strongly decreased nucleotides. To reduce their polylinker regions, constructs C trans-splicing. This shows that the evolution from and D were cut with XhoIandEcoRI,bluntendedwiththeKle- AAAAAAA to GAAGAAG, which does not affect the protein now fragment and cloned in Bluescript SK+ previously sequence, enabled the rat to produce trans-splicing of the COT digested with SacI and blunt ended with T4 DNA polymerase. pre-mRNA. Construct E was prepared by PCR taking construct B as tem- plate. The primers used were I1.f and MutESE3.r (5′- CTGACTCAAGGTATTTTTTTAGTGATTCTTC-3′). Con- MATERIALS AND METHODS struct F was prepared by the megaprimer technique taking con- Rat treatment struct A as template. Primers for the first amplification were E2.f and MutESE3.r. In the second amplification primer I2.r Groups of adult rats, weighing 200–250 g (three for each treat- was used. The constructs G, H, I and J were obtained by PCR ment) were fed for 7 days with a fat diet or a diet supplemented amplifications using genomic DNA obtained from human with diethylhexylphthalate (DEHP) as described (13), or liver. The primers were designed using the human COT cDNA injected i.p. with insulin (40 U/kg body weight) and killed at sequence AF168793 (17) and the genomic sequence 30 min. ACC005045 from the DDBJ/EMBL/GenBank database that contains the human COT genomic sequence. PCR was per- Northern blot analysis and RNase H digestion of total µ RNA formed in a total volume of 50 l with 300 ng of genomic DNA, 1 mM of each primer and Pfu polymerase. The condi- Total RNA from different samples used was extracted with tions for PCR were 94°Cfor5minfollowedby30cyclesof guanidine isothiocyanate and purified by centrifugation 94°C for 45 s, 53°C for 45 s and 72°C for 1 min. Constructs G through a CsCl cushion (14). Twenty micrograms of total liver and H were obtained with primers hE2.f (5′-GTTTCCTATT- RNA was incubated with the primer E4.r-2 (5′-CTTCTTAG- GTGATTTTATC-3′) and hI2.r (5′-TATATACTAAAACAT- CAGCTGCCAGTAG-3′) and digested with 0.8 U RNase H as TATTAAC-3′), and hE2.r (5′-CTGATTCAAGGTATTTTTT- described (5). The 32P-labeled cDNA probe used corresponds TAATG-3′)andhI1.f(5′-GCTGGTACCAAGTCCTAGA- to positions 31 to 1700 bp in the rat COT cDNA sequence (15). TATGC-3′), respectively. Construct I was obtained with prim- mRNA analysis by northern blot was performed as described ers hI1.f and MutESE4.r (5′-CTGATTCAAGGTTGTTGTT- (5). GAATGATTCTTCAAG-3′). Construct J was obtained using a megaprimer asymmetric PCR; in the first PCR primers hE2.f RT and PCR and mutESE4.r were employed to produce the megaprimer, the RT was performed in 20 µl of a mixture containing 1 µgof second PCR was performed using the megaprimer and primer total liver RNA from human, cow, mouse, pig or Cos7 cultured hI2.r. All the blunt-ended PCR fragments (E–J) were sub- monkey cells or 1 µg of liver and intestine RNA from rats. clonedinpBluescriptSK+, previously digested with Ecl136II. Random hexamers were used as primers to synthesize the For the trans-splicing experiments, construct B was linear- cDNAs. The reaction was performed as described (5). The ized with EcoRI, constructs C and D with HindIII and conditions for the PCR were 94°C for 45 s; 55°C for 45 s and constructs A and E–J with NotI and all were transcribed with 72°C for 45 s for 35 cycles. The bands were purified with T3 RNA polymerase. In vitro trans-splicing assays were Quiaex kit (Stratagene) and sequenced with an automatic carried out as described (5). The products of trans-splicing sequencer ABI prism. were loaded on a denaturing 6% polyacrylamide/7 M urea gel. Autoradiographs of the labeled bands in the gels were scanned Constructs, pre-mRNA synthesis and trans-splicing with a Molecular Dynamics laser densitometer using the reaction in vitro internal integration to determine peak areas with the Image- Constructs A and B were prepared as described (5). Construct Quant program and also with the PhosphorImager technique. A1 was also prepared as described (5) but with less polylinker The percentage inhibition was calculated from the values of region from Bluescript vector. To this end, a fragment was the integration areas of the bands. amplified with E2.f (5′-TTTTCTTACTGTGACTATAC- CATGG-3′) and I2.r (5′-CTACACTAGAAGATTATGAA- Preparation of COT minigene constructs, cell culture and CATAC-3′) and subcloned in a plasmid Bluescript SK+ previ- transfections ously cut with Ecl136II, an isoschizomer of SacI that produces A minigene containing several regions of the rat COT gene blunt ends.
Load more

Recommended publications
  • Analysis of Exonic Mutations Leading to Exon Skipping in Patients with Pyruvate Dehydrogenase E1␣ Deficiency
    0031-3998/00/4806-0748 PEDIATRIC RESEARCH Vol. 48, No. 6, 2000 Copyright © 2000 International Pediatric Research Foundation, Inc. Printed in U.S.A. Analysis of Exonic Mutations Leading to Exon Skipping in Patients with Pyruvate Dehydrogenase E1␣ Deficiency ALESSANDRA KUPPER CARDOZO, LINDA DE MEIRLEIR, INGE LIEBAERS, AND WILLY LISSENS Center for Medical Genetics [A.K.C., L.D.M., I.L., W.L.] and Pediatric Neurology [L.D.M.], University Hospital, Vrije Universiteit Brussel, 1090 Brussels, Belgium. ABSTRACT The pyruvate dehydrogenase (PDH) complex is situated at a mutations that either reverted or disrupted the wild-type pre- key position in energy metabolism and is responsible for the dicted pre-mRNA secondary structure of exon 6, we were unable conversion of pyruvate to acetyl CoA. In the literature, two to establish a correlation between the aberrant splicing and unrelated patients with a PDH complex deficiency and splicing disruption of the predicted structure. The mutagenic experiments out of exon 6 of the PDH E1␣ gene have been described, described here and the silent mutation found in one of the although intronic/exonic boundaries on either side of exon 6 patients suggest the presence of an exonic splicing enhancer in were completely normal. Analysis of exon 6 in genomic DNA of the middle region of exon 6 of the PDH E1␣ gene. (Pediatr Res these patients revealed two exonic mutations, a silent and a 48: 748–753, 2000) missense mutation. Although not experimentally demonstrated, the authors in both publications suggested that the exonic muta- tions were responsible for the exon skipping. In this work, we Abbreviations were able to demonstrate, by performing splicing experiments, ESE, exonic splicing enhancer that the two exonic mutations described in the PDH E1␣ gene mfe, minimum free energy lead to aberrant splicing.
    [Show full text]
  • Serine Arginine-Rich Protein-Dependent Suppression Of
    Serine͞arginine-rich protein-dependent suppression of exon skipping by exonic splicing enhancers El Che´ rif Ibrahim*, Thomas D. Schaal†, Klemens J. Hertel‡, Robin Reed*, and Tom Maniatis†§ *Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115; †Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138; and ‡Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697-4025 Contributed by Tom Maniatis, January 28, 2005 The 5؅ and 3؅ splice sites within an intron can, in principle, be joined mechanisms by which exon skipping is prevented. Our data to those within any other intron during pre-mRNA splicing. How- reveal that splicing to the distal 3Ј splice site is suppressed by ever, exons are joined in a strict 5؅ to 3؅ linear order in constitu- proximal exonic sequences and that SR proteins are required for tively spliced pre-mRNAs. Thus, specific mechanisms must exist to this suppression. Thus, SR protein͞exonic enhancer complexes prevent the random joining of exons. Here we report that insertion not only function in exon and splice-site recognition but also play of exon sequences into an intron can inhibit splicing to the a role in ensuring that 5Ј and 3Ј splice sites within the same intron .downstream 3؅ splice site and that this inhibition is independent of are used, thus suppressing exon skipping intron size. The exon sequences required for splicing inhibition were found to be exonic enhancer elements, and their inhibitory Materials and Methods activity requires the binding of serine͞arginine-rich splicing fac- Construction of Plasmids.
    [Show full text]
  • 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]
  • Morpholino-Mediated Exon Inclusion for SMA
    Morpholino-mediated exon inclusion for SMA Haiyan Zhou1 and Francesco Muntoni1* 1The Dubowitz Neuromuscular Centre, Molecular Neurosciences Session, Developmental Neurosciences Programme, Great Ormond Street Institute of Child Health, University College London. 30 Guilford Street, London WC1N 1EH. United Kingdom. E-mail: [email protected] Abstract The application of antisense oligonucleotides (AONs) to modify pre-messenger RNA splicing has great potential for treating genetic diseases. The strategies used to redirect splicing for therapeutic purpose involve the use of AONs complementary to splice motifs, enhancer or silencer sequences. AONs to block intronic splicing silencer motifs can efficiently augment exon 7 inclusion in survival motor neuron 2 (SMN2) gene and have demonstrated robust therapeutic effects in both pre-clinical studies and clinical trials in spinal muscular atrophy (SMA), which lead to a recently approved drug. AONs with phosphoroamidate morpholino (PMO) backbone have shown target engagement with restoration of the defective protein in Duchenne muscular dystrophy (DMD) and their safety profile lead to a recent conditional approval for one DMD PMO drug. PMO AONs are also effective in correcting SMN2 exon 7 splicing and rescuing SMA transgenic mice. Here we provide the details of methods that our lab has used to evaluate PMO-mediated SMN2 exon 7 inclusion in the in vivo studies conducted in SMA transgenic mice. The methods comprise mouse experiment procedures, assessment of PMOs on exon 7 inclusion at RNA levels by reverse transcription (RT-) PCR and quantitative real-time PCR. In addition, we present methodology for protein quantification using western blot in mouse tissues, on neuropathology assessment of skeletal muscle (muscle pathology and neuromuscular junction staining) as well as behaviour test in the SMA mice (righting reflex).
    [Show full text]
  • Distribution of SR Protein Exonic Splicing Enhancer Motifs in Human Protein-Coding Genes Jinhua Wang†, Philip J
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central Nucleic Acids Research, 2005, Vol. 33, No. 16 5053–5062 doi:10.1093/nar/gki810 Distribution of SR protein exonic splicing enhancer motifs in human protein-coding genes Jinhua Wang†, Philip J. Smith†, Adrian R. Krainer and Michael Q. Zhang* Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA Received April 12, 2005; Revised July 22, 2005; Accepted August 16, 2005 ABSTRACT sequences that conform to the splice-site consensus motifs at least as well as those utilized by many true exons (2). Exonic splicing enhancers (ESEs) are pre-mRNA cis- The additional information required for exon definition is con- acting elements required for splice-site recognition. tained at least partly in cis-acting regulatory enhancer and We previously developed a web-based program called silencer sequences (3). ESEfinder that scores any sequence for the presence Exonic splicing enhancers (ESEs) participate in both alter- of ESE motifs recognized by the human SR proteins native and constitutive splicing, and many of them act as SF2/ASF, SRp40, SRp55 and SC35 (http://rulai.cshl. binding sites for members of the SR protein family (4,5). edu/tools/ESE/). Using ESEfinder, we have under- The SR proteins are a family of related proteins that share taken a large-scale analysis of ESE motif distribution a conserved domain structure. They have one or two copies of in human protein-coding genes. Significantly higher an RNA-recognition motif (RRM) followed by a C-terminal frequencies of ESE motifs were observed in consti- domain that is highly enriched in arginine/serine dipeptides (RS domain) (6).
    [Show full text]
  • 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.
    [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]
  • RESCUE-ESE Identifies Candidate Exonic Splicing Enhancers in Vertebrate Exons William G
    Nucleic Acids Research, 2004, Vol. 32, Web Server issue W187–W190 DOI: 10.1093/nar/gkh393 RESCUE-ESE identifies candidate exonic splicing enhancers in vertebrate exons William G. Fairbrother1,2, Gene W. Yeo2, Rufang Yeh3, Paul Goldstein4, Matthew Mawson2, Phillip A. Sharp1,2,5 and Christopher B. Burge2,* 1Center for Cancer Research, 2Department of Biology and 5McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA, 3Department of Epidemiology and Biostatistics, University of California San Francisco, 500 Parnassus Avenue Box 0560, San Francisco, CA 94143-0560, USA and 446 Glenbrook Road, W. Hartford, CT 06107, USA Downloaded from Received February 13, 2004; Revised and Accepted March 24, 2004 ABSTRACT by proteins of the SR family, which function by recruiting components of the core splicing machinery to nearby splice A typical gene contains two levels of information: a sites (6) or by counteracting the effects of nearby silencing sequence that encodes a particular protein and a host elements (7). http://nar.oxfordjournals.org/ of other signals that are necessary for the correct A variety of selection schemes have been used to determine expression of the transcript. While much attention which sequences are capable of functioning as ESEs (2–5). has been focused on the effects of sequence variation These SELEX methods start with a complex pool of random on the amino acid sequence, variations that disrupt sequence and invoke an iterative selection–amplification pro- gene processing signals can dramatically impact tocol which progressively enriches the fraction of molecules in gene function. A variation that disrupts an exonic the total pool that possess ESE activity.
    [Show full text]
  • Evaluating the Performance of in Silico Predictive Models on Detecting Splice-Altering Variants
    Dominican Scholar Graduate Master's Theses, Capstones, and Culminating Projects Student Scholarship 5-2016 Evaluating the Performance of in Silico Predictive Models on Detecting Splice-altering Variants Erica Cayton Dominican University of California https://doi.org/10.33015/dominican.edu/2016.bio.05 Survey: Let us know how this paper benefits you. Recommended Citation Cayton, Erica, "Evaluating the Performance of in Silico Predictive Models on Detecting Splice- altering Variants" (2016). Graduate Master's Theses, Capstones, and Culminating Projects. 226. https://doi.org/10.33015/dominican.edu/2016.bio.05 This Master's Thesis is brought to you for free and open access by the Student Scholarship at Dominican Scholar. It has been accepted for inclusion in Graduate Master's Theses, Capstones, and Culminating Projects by an authorized administrator of Dominican Scholar. For more information, please contact [email protected]. Evaluating the performance of in silico predictive models on detecting splice-altering variants By Erica C. Cayton A culminating thesis submitted to the faculty of Dominican University of California and BioMarin Pharmaceutical Inc. in partial fulfillment of the requirements for the degree of Master of Science in Biology San Rafael, California May 2015 Copyright 2016, by Erica C. Cayton All Rights Reserved ii Contents 1Introduction................................ 1 1.1 Splicing and Disease ....................... 5 1.2 The Molecular Biology of Splicing ................ 10 1.3 Splicing in Evolutionary Context ................ 13 2ReviewofPredictionMethods...................... 16 3DataandMethods............................ 23 3.1 Evaluated Predictors ....................... 23 3.2 Evaluation Data Sets ....................... 23 3.3 Using ROC Curves to Quantify Predictor Performance .... 24 4Results................................... 28 4.1 Performance on Exonic Missense Data Set ..........
    [Show full text]
  • Alternatively Spliced Genes
    1 Alternatively Spliced Genes Jane Y. Wu1,2,LiyaYuan1 and Necat Havlioglu1 1Washington University School of Medicine, St. Louis, MO, USA 2John F. Kennedy Center for Research on Human Development, Vanderbilt University Medical Center, Nashville, TN, USA 1 Pre-mRNA Splicing and Splicing Machinery 3 1.1 Splicing Machinery: Spliceosome 3 1.2 Splicing Signals 4 1.3 Spliceosomal UsnRNP Biogenesis 12 1.4 Spliceosome Assembly 13 1.5 Biochemical Mechanisms of pre-mRNA Splicing 17 2 Alternative pre-mRNA Splicing 17 2.1 Alternative Splicing and its Role in Regulating Gene Activities and Generating Genetic Diversity 17 2.1.1 Different Patterns of Alternative Splicing 17 2.1.2 Alternative Splicing and Genetic Diversity 18 2.2 Mechanisms Underlying Alternative Splicing Regulation 19 2.2.1 Splicing Signals and Splicing Regulatory Elements 20 2.2.2 Trans-acting Splicing Regulators 23 2.3 Tissue-specific and Developmentally Regulated Alternative Splicing 26 2.4 Regulation of Alternative Splicing in Response to Extracellular Stimuli 27 3 Pre-mRNA Splicing and Human Diseases 28 3.1 Splicing Defects in Human Diseases 28 3.2 Molecular Mechanisms Underlying Splicing Defects Associated with Disease 33 4 Perspectives on Diagnosis and Treatment of Diseases Caused by pre-mRNA Splicing Defects 36 4.1 Diagnosis of Human Diseases Caused by Splicing Defects 36 2 Alternatively Spliced Genes 4.2 Potential Therapeutic Approaches 37 4.2.1 Oligonucleotide-based Approaches: Antisense, RNAi, and Chimeric Molecules 37 4.2.2 Ribozymes 37 4.2.3 SMaRT 38 4.2.4 Chemical Compounds 38 5 Concluding Remarks 38 Acknowledgment 39 Bibliography 39 Books and Reviews 39 Keywords Pre-mRNA Nascent transcripts that are precursors of mature messenger RNAs.
    [Show full text]
  • A New Regulatory Protein, KSRP, Mediates Exon Inclusion Through an Intronic Splicing Enhancer
    Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press A new regulatory protein, KSRP, mediates exon inclusion through an intronic splicing enhancer Hosung Min, 1 Christoph W. Turck, 4 Julia M. Nikolic, 3 and Douglas L. Black 1-3's 1Molecular Biology Institute, 2Department of Microbiology and Molecular Genetics, and 3Howard Hughes Medical Institute, University of California at Los Angeles, Los Angeles, California 90095-1662 USA; 4Howard Hughes Medical Institute, Department of Medicine and Cardiovascular Research Institute, University of California at San Francisco, San Francisco, California 94143-0724 USA We have purified and cloned a new splicing factor, KSRP. KSRP is a component of a multiprotein complex that binds specifically to an intronic splicing enhancer element downstream of the neuron-specific c-src N1 exon. This 75-kD protein induces the assembly of five other proteins, including the heterogeneous nuclear ribonucleoprotein F, onto the splicing enhancer. The sequence of the KSRP cDNA indicates that the protein contains four K homology RNA-binding domains and an unusual carboxy-terminal domain. KSRP is similar to two proteins, FUSE-binding protein and P-element somatic inhibitor. KSRP is expressed in both neural and non-neural cell lines, although it is present at higher levels in neural cells. Antibodies specific for KSRP inhibit the splicing of the N1 exon in vitro. Moreover, this inhibition of N1 splicing can be rescued by the addition of purified KSRP. KSRP is likely to regulate splicing from a number of intronic splicing enhancer sequences. [Key Words: Alternative splicing; regulatory protein; KSRP; intronic splicing enhancer; RNA-binding protein] Received October 22, 1996; revised version accepted March 4, 1997.
    [Show full text]
  • Mutational Bias and the Protein Code Shape the Evolution of Splicing Enhancers
    ARTICLE https://doi.org/10.1038/s41467-020-16673-z OPEN Mutational bias and the protein code shape the evolution of splicing enhancers Stephen Rong 1,2, Luke Buerer1, Christy L. Rhine3, Jing Wang3, Kamil J. Cygan 1,3 & ✉ William G. Fairbrother1,3,4 Exonic splicing enhancers (ESEs) are enriched in exons relative to introns and bind splicing activators. This study considers a fundamental question of co-evolution: How did ESE motifs 1234567890():,; become enriched in exons prior to the evolution of ESE recognition? We hypothesize that the high exon to intron motif ratios necessary for ESE function were created by mutational bias coupled with purifying selection on the protein code. These two forces retain certain coding motifs in exons while passively depleting them from introns. Through the use of simulations, genomic analyses, and high throughput splicing assays, we confirm the key predictions of this hypothesis, including an overlap between protein and splicing information in ESEs. We discuss the implications of mutational bias as an evolutionary driver in other cis-regulatory systems. 1 Center for Computational Molecular Biology, Brown University, Providence, RI 02912, USA. 2 Ecology and Evolutionary Biology, Brown University, Providence, RI 02912, USA. 3 Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA. 4 Hassenfeld Child Health ✉ Innovation Institute of Brown University, Providence, RI 02912, USA. email: [email protected] NATURE COMMUNICATIONS | (2020) 11:2845 | https://doi.org/10.1038/s41467-020-16673-z | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16673-z plicing refers to the process of removing long intervening depleted from the genome.
    [Show full text]