DOCSLIB.ORG

Mechanisms and Impact of Post-Transcriptional Exon Shuffling

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mechanisms and Impact of Post-Transcriptional Exon Shuffling Mechanisms and impact of Post-Transcriptional Exon Shuffling (PTES) Ginikachukwu Osagie Izuogu Doctor of Philosophy Institute of Genetic Medicine Newcastle University September 2016 i ABSTRACT Most eukaryotic genes undergo splicing to remove introns and join exons sequentially to produce protein-coding or non-coding transcripts. Post-transcriptional Exon Shuffling (PTES) describes a new class of RNA molecules, characterized by exon order different from the underlying genomic context. PTES can result in linear and circular RNA (circRNA) molecules and enhance the complexity of transcriptomes. Prior to my studies, I developed PTESFinder, a computational tool for PTES identification from high-throughput RNAseq data. As various sources of artefacts (including pseudogenes, template-switching and others) can confound PTES identification, I first assessed the effectiveness of filters within PTESFinder devised to systematically exclude artefacts. When compared to 4 published methods, PTESFinder achieves the highest specificity (~0.99) and comparable sensitivity (~0.85). To define sub-cellular distribution of PTES, I performed in silico analyses of data from various cellular compartments and revealed diverse populations of PTES in nuclei and enrichment in cytosol of various cell lines. Identification of PTES from chromatin-associated RNAseq data and an assessment of co-transcriptional splicing, established that PTES may occur during transcription. To assess if PTES contribute to the proteome, I analyzed sucrose-gradient fractionated data from HEK293, treated with arsenite to induce translational arrest and dislodge ribosomes. My results showed no effect of arsenite treatment on ribosome occupancy within PTES transcripts, indicating that these transcripts are not generally bound by polysomes and do not contribute to the proteome. To investigate the impact of differential degradation on expression levels of linear and circRNAs, I analyzed the PTES population within RNAseq data of anucleate cells and established that most PTES transcripts are circular and are enriched in platelets 17-to-188-fold relative to nucleated tissues. For some genes, only reads from circRNA exons were detectable, suggesting that platelets have lost >90% of their progenitor mRNAs, consistent with time- dependent degradation of platelets transcriptomes. However, some circRNAs exhibit read density patterns suggestive of miRNA induced degradation. Finally, a linear PTES from RMST locus has been implicated in pluripotency maintenance using limited RNAseq data from human embryonic stem cells (hESC). To identify other PTES transcripts with similar expression patterns, I analyzed RNAseq data from H9 ESC differentiation series. Statistical analyses of PTES transcripts identified during cellular differentiation established that PTES expression changes track with that of cognate linear transcripts and accumulate upon differentiation. Contrary to previous reports, the dominant transcript from RMST is circular and increases in abundance during differentiation. Functional Abstract ii analyses demonstrating the role of RMST in pluripotency maintenance had targeted exons within the predicted circRNA, suggesting previously unreported functional relevance for circRNAs. Abstract iii Declaration I, Osagie Izuogu, declare that no material documented in this thesis has been submitted in support of a degree or other qualification in this or any other university. This thesis represents my own work and any collaborative work is acknowledged where necessary. Osagie Izuogu Declaration iv Dedication This thesis is dedicated to Denise, my wife; Tiana, Dante and Luca, my children. Thank you all for your unwavering support and understanding throughout my research. I love you all. Dedication v Acknowledgements Enormous gratitude to Dr. Mike Jackson and Dr. Mauro Santibanez-Koref for the opportunity to undertake my research under their supervision. I am particularly grateful for your guidance, support and patience, and for being available for all formal and informal discussions. I sincerely look forward to future opportunities to work with you, thank you. To Dr. Alhassan, thank you for your guidance and assistance with in vitro analyses, I appreciate your help. To Prof. David Elliott and members of Elliott’s lab, thank you for your support and for providing answers to my numerous questions. I would like to thank current and former members of the statistical genetics group (IGM), especially, Dr. Miossecc, Dr. Griffin, Dr. Ayers, Dr. Xu, Dr. Ainsworth and Dr. Howey. I thank you all for your words of advice, encouragement and impromptu tutorials. Our informal meetings helped shape my thoughts. Special thanks to my collaborators - Prof Lako (Newcastle University, UK), Dr. Ghevaert & colleagues (Cambridge University, UK) - and my panel members - Prof. Heather Cordell, Dr. Andreas Werner and Dr. Venables; I thoroughly enjoyed working on this project and my research benefitted from your expertise, words of encouragement and guidance, thank you. To Dr. Harry Mountain (Staffordshire University, UK) and Ada Izuogu (University of Toledo, Ohio), thank you both for proofreading my thesis and your valuable comments. I am grateful to my family and friends, for their unconditional love and support throughout my study, thank you all. I acknowledge the Biotechnology and Biological Sciences Research Council for funding my research. Ultimately, I thank God for his mercies. Acknowledgements vi vii Table of Contents List of Figures 14 List of Tables 16 List of Abbreviations 17 Chapter 1: Introduction 19 1.1 Transcriptome Diversity in Humans 19 1.2 Major Sources of Transcriptome Diversity 21 1.2.1 Alternative Splicing 21 1.2.2 Transcription of 'Junk DNA' 23 1.2.3 Chimeric Transcripts 26 1.3 Post-Transcriptional Exon Shuffling (PTES) 28 1.3.1 Mechanisms of PTES Formation 31 1.3.2 Regulation of PTES Formation 33 1.3.3 In vitro methods for identification of PTES 35 1.3.4 Approaches to PTES in silico identification 37 1.3.5 Sources of known artefacts that confound in silico PTES identification 41 1.3.6 PTESFinder: a computational tool for PTES identification 43 1.4 Project Aims 46 Chapter 2: Materials & Methods 47 2.1 Cell lines 47 2.2 Sample Preparation 47 2.2.1 Tissue culture of HEK293 and DAOY cell lines 47 2.2.2 Differentiation of H9 ESC 47 2.2.3 Human tissues and blood samples from healthy donors 48 2.2.4 RNA Isolation and cDNA synthesis 48 Table of Contents 8 2.2.5 RNase R digestion 49 2.3 In vitro PTES confirmation, visualization and quantification 49 2.3.1 Primer design 49 2.3.2 Polymerase Chain Reaction (PCR) 49 2.3.3 Agarose Gel electrophoresis 50 2.3.4 Quantitative PCR (qPCR) 50 2.4 Public RNAseq datasets 50 2.4.1 Human Fibroblasts and Leukocytes data 50 2.4.2 ENCODE sub-cellular RNAseq data 51 2.4.3 Sucrose-gradient fractionated RNAseq data from HEK293 51 2.4.4 RNAseq data from human tissues and anucleate cells 51 2.5 RNAseq data generation 52 2.5.1 High-throughput RNA sequencing 52 2.5.2 Generating simulated RNAseq data 53 2.5.3 Sub-sampling of RNAseq data 54 2.6 Computational Methods 54 2.6.1 Sequence Quality check 54 2.6.2 PTES identification 54 2.6.3 RNAseq analysis 55 2.6.4 Definition and derivation of metrics 56 2.6.5 Statistical analysis of PTES abundance 58 2.6.6 Custom scripts 58 Chapter 3: Assessment of Computational PTES identification Methods 59 3.1 Introduction 59 3.1.1 Existing PTES identification tools do not specifically exclude all sources of artefacts 59 Table of Contents 9 3.1.2 Choice of aligner and aligner-specific parameters may impact reproducibility of PTES predictions 60 3.1.3 PTESFinder is equipped with filters that systematically exclude sources of artefacts 61 3.2 Aims 63 3.3 Results 64 3.3.1 Filters Target Overlapping Populations Of Reads 64 3.3.2 Reads Excluded By Specific Filters Have Different Origins 66 3.3.3 PID Has Greater Impact Than JSpan 69 3.3.4 Effect of Aligner Specific Parameter and PTESFinder Performance 70 3.3.5 Comparison of PTES identification methods 72 3.3.6 Assessment of Annotation-Free Identification Methods 77 3.4 Discussion 81 3.5 Conclusion 82 Chapter 4: Sub-Cellular Distribution of PTES transcripts 84 4.1 Introduction 84 4.1.1 Spliceosomal proteins aid nucleo-cytoplasmic mRNA export 84 4.1.2 Co- or Post-Transcriptional Exon Shuffling? 86 4.1.3 Profiling transcripts undergoing translation 87 4.2 Aims 89 4.3 Results 90 4.3.1 Variety of PTES events observed in the nucleus 91 4.3.2 snoRNA-PTES transcripts are likely artefacts 92 4.3.3 PTES transcripts are enriched in the cytosol 98 4.3.4 Incompletely processed circRNAs enriched in the nucleus 101 4.3.5 Quantitative analysis of chromatin-associated PTES transcripts 103 Table of Contents 10 4.3.6 Do PTES transcripts contribute to the proteome? 107 4.4 Discussion 109 4.5 Conclusion 112 Chapter 5: PTES transcripts in anucleate cells 113 5.1 Introduction 113 5.1.1 Platelets have complex transcriptomes 113 5.1.2 Platelets transcriptomes vary between human donors 114 5.2 Aims 116 5.3 Results 117 5.3.1 Most PTES transcripts identified in platelets are circular 118 5.3.2 CircRNAs are enriched in anucleate cells and expand the growing catalog of PTES transcripts 120 5.3.3 Reads from circRNA producing exons are enriched in Platelets 124 5.3.4 circRNA abundance in Platelets is due to decay of linear transcripts 128 5.3.5 RNA secondary structure, GC content and miRNA binding sites may contribute to circRNA stability 130 5.4 Discussion 138 5.5 Conclusion 140 Chapter 6: PTES Events in development 141 6.1 Introduction
Load more

Recommended publications
  • "Evolutionary Emergence of Genes Through Retrotransposition"
    Evolutionary Emergence of Advanced article Genes Through Article Contents . Introduction Retrotransposition . Gene Alteration Following Retrotransposon Insertion . Retrotransposon Recruitment by Host Genome . Retrotransposon-mediated Gene Duplication Richard Cordaux, University of Poitiers, Poitiers, France . Conclusion Mark A Batzer, Department of Biological Sciences, Louisiana State University, Baton Rouge, doi: 10.1002/9780470015902.a0020783 Louisiana, USA Variation in the number of genes among species indicates that new genes are continuously generated over evolutionary times. Evidence is accumulating that transposable elements, including retrotransposons (which account for about 90% of all transposable elements inserted in primate genomes), are potent mediators of new gene origination. Retrotransposons have fostered genetic innovation during human and primate evolution through: (i) alteration of structure and/or expression of pre-existing genes following their insertion, (ii) recruitment (or domestication) of their coding sequence by the host genome and (iii) their ability to mediate gene duplication via ectopic recombination, sequence transduction and gene retrotransposition. Introduction genes, respectively, and de novo origination from previ- ously noncoding genomic sequence. Genome sequencing Variation in the number of genes among species indicates projects have also highlighted that new gene structures can that new genes are continuously generated over evolution- arise as a result of the activity of transposable elements ary times. Although the emergence of new genes and (TEs), which are mobile genetic units or ‘jumping genes’ functions is of central importance to the evolution of that have been bombarding the genomes of most species species, studies on the formation of genetic innovations during evolution. For example, there are over three million have only recently become possible.
    [Show full text]
  • Identification and Characterization of Breakpoints and Mutations on Drosophila Melanogaster Balancer Chromosomes
    G3: Genes|Genomes|Genetics Early Online, published on September 24, 2020 as doi:10.1534/g3.120.401559 1 Identification and characterization of breakpoints and mutations on Drosophila 2 melanogaster balancer chromosomes 3 Danny E. Miller*, Lily Kahsai†, Kasun Buddika†, Michael J. Dixon†, Bernard Y. Kim‡, Brian R. 4 Calvi†, Nicholas S. Sokol†, R. Scott Hawley§** and Kevin R. Cook† 5 *Department of Pediatrics, Division of Genetic Medicine, University of Washington, and Seattle 6 Children's Hospital, Seattle, WA, 98105 7 †Department of Biology, Indiana University, Bloomington, IN 47405 8 ‡Department of Biology, Stanford University, Stanford, CA 94305 9 §Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 10 Kansas City, KS 66160 11 **Stowers Institute for Medical Research, Kansas City, MO 64110 12 ORCIDs: 0000-0001-6096-8601 (D.E.M.), 0000-0003-3429-445X (L.K.), 0000-0002-9872-4765 13 (K.B.), 0000-0003-0208-0641 (M.J.D.), 0000-0002-5025-1292 (B.Y.K.), 0000-0001-5304-0047 14 (B.R.C.), 0000-0002-2768-4102 (N.S.S.), 0000-0002-6478-0494 (R.S.H.), 0000-0001-9260- 15 364X (K.R.C.) 1 © The Author(s) 2020. Published by the Genetics Society of America. 16 Running title: Balancer breakpoints and phenotypes 17 18 Keywords: balancer chromosomes, chromosomal inversions, p53, Ankyrin 2, 19 Fucosyltransferase A 20 21 Corresponding author: Kevin R. Cook, Department of Biology, Indiana University, 1001 E. 22 Third St., Bloomington, IN 47405, 812-856-1213, [email protected] 23 2 24 ABSTRACT 25 Balancers are rearranged chromosomes used in Drosophila melanogaster to maintain 26 deleterious mutations in stable populations, preserve sets of linked genetic elements and 27 construct complex experimental stocks.
    [Show full text]
  • Origins of New Genes: Exon Shuffling
    OriginsOrigins ofof NewNew Genes:Genes: ExonExon ShufflingShuffling By Carl Hillstrom 1 • The talk is about how the shuffling of exons can give rise to new genes. 2 MerriamMerriam--WebsterWebster OnlineOnline DictionaryDictionary Main Entry: ex·on Pronunciation: 'ek-"sän Function: noun : a polynucleotide sequence in a nucleic acid that codes information for protein synthesis and that is copied and spliced together with other such sequences to form messenger RNA -- compare INTRON http://www.m-w.com/dictionary/exon 3 ExonExon shufflingshuffling Recombination, exclusion, or duplications of exons can drive the evolution of new genes. The general idea of exon shuffling is typically attributed to Walter Gilbert (e.g. Long et al. 2003) The definition of exon shuffling used in this presentation encompass: --exon assumes a new function after it has been moved --exon retains its original function after it has been moved 4 • So what is exon shuffling? • It is basically the idea that recombination or exclusion of exons can drive the evolution of new genes. “Recombination, exclusion, or duplications of exons can drive the evolution of new genes.” –this is a very general definition that I have adopted for the purpose of this presentation. • The definition of exon shuffling used in this presentation encompass: • --exon assumes a new function after it has been moved • --exon retains its original function after it has been moved • there is disagreement whether exon shuffling applies to both of these definition—for simplicity, I will use the concept of exon shuffling as if it applies to both of these definitions 5 OutlineOutline ofof aa typicaltypical antibodyantibody Alberts et al.
    [Show full text]
  • L1 Retrotransposition Is a Common Feature of Mammalian Hepatocarcinogenesis
    Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Research L1 retrotransposition is a common feature of mammalian hepatocarcinogenesis Stephanie N. Schauer,1,12 Patricia E. Carreira,1,12 Ruchi Shukla,2,12 Daniel J. Gerhardt,1,3 Patricia Gerdes,1 Francisco J. Sanchez-Luque,1,4 Paola Nicoli,5 Michaela Kindlova,1 Serena Ghisletti,6 Alexandre Dos Santos,7,8 Delphine Rapoud,7,8 Didier Samuel,7,8 Jamila Faivre,7,8,9 Adam D. Ewing,1 Sandra R. Richardson,1 and Geoffrey J. Faulkner1,10,11 1Mater Research Institute–University of Queensland, Woolloongabba, QLD 4102, Australia; 2Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne NE1 7RU, United Kingdom; 3Invenra, Incorporated, Madison, Wisconsin 53719, USA; 4Department of Genomic Medicine, GENYO, Centre for Genomics and Oncological Research: Pfizer-University of Granada- Andalusian Regional Government, PTS Granada, 18016 Granada, Spain; 5Department of Experimental Oncology, European Institute of Oncology, 20146 Milan, Italy; 6Humanitas Clinical and Research Center, 20089 Milan, Italy; 7INSERM, U1193, Paul- Brousse University Hospital, Hepatobiliary Centre, Villejuif 94800, France; 8Université Paris-Sud, Faculté de Médecine, Villejuif 94800, France; 9Assistance Publique-Hôpitaux de Paris (AP-HP), Pôle de Biologie Médicale, Paul-Brousse University Hospital, Villejuif 94800, France; 10School of Biomedical Sciences, University of Queensland, Brisbane, QLD 4072, Australia; 11Queensland Brain Institute, University of Queensland, Brisbane, QLD 4072, Australia The retrotransposon Long Interspersed Element 1 (LINE-1 or L1) is a continuing source of germline and somatic mutagenesis in mammals. Deregulated L1 activity is a hallmark of cancer, and L1 mutagenesis has been described in numerous human malignancies.
    [Show full text]
  • INTRINSIC and EXTRINSIC EVOLUTION of HELITRONS in FLOWERING PLANT GENOMES by LIXING YANG (Under the Direction of Jeffrey L. Benn
    INTRINSIC AND EXTRINSIC EVOLUTION OF HELITRONS IN FLOWERING PLANT GENOMES by LIXING YANG (Under the Direction of Jeffrey L. Bennetzen) ABSTRACT Helitrons are a recently discovered class of eukaryotic transposable elements that are believed to transpose by a rolling circle mechanism. Because Helitrons frequently acquire and fuse fragments of multiple genes, they may be major contributors to the creation of novel genes by exon shuffling. This dissertation provides a comprehensive study of Helitrons in flowering plant genomes including their identification in several different genomes, their structural features, and their roles in genome evolution. Helitrons can be difficult to identify because they have few and tiny conserved structures. We developed a new approach to effectively identify Helitrons (tested in Arabidopsis and nematodes) and further refined and demonstrated this approach on a few completely sequenced plant genomes (Medicago, rice and sorghum). We discovered a large number of new elements and new element families, and also a few new Helitron characteristics. We identified initiation and termination bypass events that led to new 5′ or 3′ ends that created newly active families and subfamilies of Helitrons. We found that Helitrons preferentially insert into AT-rich regions, that they prefer to insert near other Helitrons, and that the predicted hairpins near their 3′ ends would have high predicted melting temperatures. Maize Helitrons are known to acquire gene fragments frequently. With the completion of the maize genome sequencing project this year, we were able to perform a large-scale search for Helitrons in the maize genome. We discovered 1930 intact elements in the maize genome, and were able to predict more than 20,000 total elements that account for just over 2% of the sequence assembly.
    [Show full text]
  • Living Organisms Author Their Read-Write Genomes in Evolution
    biology Review Living Organisms Author Their Read-Write Genomes in Evolution James A. Shapiro ID Department of Biochemistry and Molecular Biology, University of Chicago GCIS W123B, 979 E. 57th Street, Chicago, IL 60637, USA; [email protected]; Tel.: +1-773-702-1625 Academic Editor: Andrés Moya Received: 23 August 2017; Accepted: 28 November 2017; Published: 6 December 2017 Abstract: Evolutionary variations generating phenotypic adaptations and novel taxa resulted from complex cellular activities altering genome content and expression: (i) Symbiogenetic cell mergers producing the mitochondrion-bearing ancestor of eukaryotes and chloroplast-bearing ancestors of photosynthetic eukaryotes; (ii) interspecific hybridizations and genome doublings generating new species and adaptive radiations of higher plants and animals; and, (iii) interspecific horizontal DNA transfer encoding virtually all of the cellular functions between organisms and their viruses in all domains of life. Consequently, assuming that evolutionary processes occur in isolated genomes of individual species has become an unrealistic abstraction. Adaptive variations also involved natural genetic engineering of mobile DNA elements to rewire regulatory networks. In the most highly evolved organisms, biological complexity scales with “non-coding” DNA content more closely than with protein-coding capacity. Coincidentally, we have learned how so-called “non-coding” RNAs that are rich in repetitive mobile DNA sequences are key regulators of complex phenotypes. Both biotic and abiotic ecological challenges serve as triggers for episodes of elevated genome change. The intersections of cell activities, biosphere interactions, horizontal DNA transfers, and non-random Read-Write genome modifications by natural genetic engineering provide a rich molecular and biological foundation for understanding how ecological disruptions can stimulate productive, often abrupt, evolutionary transformations.
    [Show full text]
  • Alternative Splicing and Evolution: Diversification, Exon Definition and Function
    Nature Reviews Genetics | AOP, published online 8 April 2010; doi:10.1038/nrg2776 REVIEWS Alternative splicing and evolution: diversification, exon definition and function Hadas Keren, Galit Lev-Maor and Gil Ast Abstract | Over the past decade, it has been shown that alternative splicing (AS) is a major mechanism for the enhancement of transcriptome and proteome diversity, particularly in mammals. Splicing can be found in species from bacteria to humans, but its prevalence and characteristics vary considerably. Evolutionary studies are helping to address questions that are fundamental to understanding this important process: how and when did AS evolve? Which AS events are functional? What are the evolutionary forces that shaped, and continue to shape, AS? And what determines whether an exon is spliced in a constitutive or alternative manner? In this Review, we summarize the current knowledge of AS and evolution and provide insights into some of these unresolved questions. Spliceosome Splicing of precursor mRNA (pre-mRNA) is a crucial regulated at many different levels, often in a tissue- or A ribonucleoprotein complex regulatory stage in the pathway of gene expression: developmental stage-specific manner. At a basic level, that is involved in splicing introns are removed and exons are ligated to form regulation includes splice-site recognition by the of nuclear precursor mRNA mRNA. The inclusion of different exons in mRNA — spliceosome, which is mediated by various proteins. (pre-mRNA). It is composed of five small nuclear alternative splicing (AS) — results in the generation of Additional regulatory levels include environmental ribonucleoproteins (snRNPs) different isoforms from a single gene and is the basis changes that affect splice-site choice and the relationship and more than 50 non-snRNPs, for the discrepancy between the estimated 24,000 among transcription by RNA polymerase II (RNAPII), which recognize and assemble protein-coding genes in the human genome and the nucleosome occupancy and splicing7–10.
    [Show full text]
  • Exon Shuffling Played a Decisive Role in the Evolution of the Genetic Toolkit for the Multicellular Body Plan of Metazoa
    G C A T T A C G G C A T genes Opinion Exon Shuffling Played a Decisive Role in the Evolution of the Genetic Toolkit for the Multicellular Body Plan of Metazoa Laszlo Patthy Institute of Enzymology, Research Centre for Natural Sciences, H-1117 Budapest, Hungary; [email protected]; Tel.: +36-13826751 Abstract: Division of labor and establishment of the spatial pattern of different cell types of multicellu- lar organisms require cell type-specific transcription factor modules that control cellular phenotypes and proteins that mediate the interactions of cells with other cells. Recent studies indicate that, although constituent protein domains of numerous components of the genetic toolkit of the mul- ticellular body plan of Metazoa were present in the unicellular ancestor of animals, the repertoire of multidomain proteins that are indispensable for the arrangement of distinct body parts in a reproducible manner evolved only in Metazoa. We have shown that the majority of the multidomain proteins involved in cell–cell and cell–matrix interactions of Metazoa have been assembled by exon shuffling, but there is no evidence for a similar role of exon shuffling in the evolution of proteins of metazoan transcription factor modules. A possible explanation for this difference in the intracellular and intercellular toolkits is that evolution of the transcription factor modules preceded the burst of exon shuffling that led to the creation of the proteins controlling spatial patterning in Metazoa. This explanation is in harmony with the temporal-to-spatial transition hypothesis of multicellu- larity that proposes that cell differentiation may have predated spatial segregation of cell types in animal ancestors.
    [Show full text]
  • Exon-Trapping Mediated by the Human Retrotransposon SVA
    Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Exon-trapping mediated by the human retrotransposon SVA Dustin C. Hancks1,3, Adam D. Ewing1,3, Jesse E. Chen1, Katsushi Tokunaga2, and Haig H. Kazazian Jr1,4 1 Department of Genetics, University of Pennsylvania 2Department of Human Genetics, Graduate School of Medicine, University of Tokyo, Tokyo, Japan 3 These authors contributed equally. Running title: Exon-trapping mediated by the human retrotransposon SVA Key Words: SVA, retrotransposition, alternative splicing 4Corresponding author: Haig H. Kazazian Jr., Correspondence to: Haig H. Kazazian Jr., Department of Genetics, University of Pennsylvania, School of Medicine, 564 Clinical Research Building, 415 Curie Blvd., Philadelphia, PA 19104-6145 Tel: 215-898-3582 E-mail: [email protected] 1 Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Abstract Although most human retrotransposons are inactive, both inactive and active retrotransposons drive genome evolution and may influence transcription through various mechanisms. In humans, three retrotransposon families are still active, but one of these, SVA, remains mysterious. Here we report the identification of a new subfamily of SVA, which apparently formed after an alternative splicing event, where the first exon of the MAST2 gene spliced into an intronic SVA and subsequently retrotransposed. Additional examples of SVA retrotransposing upstream exons due to splicing into SVA were also identified in other primate genomes. After molecular and computational experiments, we found a number of functional 3’ splice sites within many different transcribed SVAs across the human and chimpanzee genomes.
    [Show full text]
  • Discovery of RNA Splicing and Genes in Pieces PERSPECTIVE Arnold J
    PERSPECTIVE Discovery of RNA splicing and genes in pieces PERSPECTIVE Arnold J. Berka,1 Edited by Inder M. Verma, The Salk Institute for Biological Studies, La Jolla, CA, and approved December 29, 2015 (received for review December 18, 2015) The Problem: Short-Lived Heterogeneous of unlabeled nucleoside to the culture medium during Nuclear RNA the chase period. Consequently, a clean chase could During the 1960s and 1970s, there was a major co- not be achieved, and newly synthesized hnRNAs nundrum in eukaryotic molecular biology regarding continued to be labeled as labeled mRNAs first the synthesis of mRNA in animal cells. Pulse and pulse- appeared in the cytoplasm. Also, the small fraction of chase radio-labeling experiments with tritiated uridine pulse-labeled nuclear RNA converted to more stable had shown that a large fraction of nuclear RNA was mRNAs, only 5–10%, added to the difficulty of proving degraded very rapidly after synthesis, whereas only a a precursor product relationship from pulse-chase small fraction of the initially synthesized RNA was labeling experiments. exported from the nucleus to the cytoplasm where it The synthesis and processing of ribosomal RNAs functions as far more stable mRNAs (1, 2). This short- was much easier to analyze than processing of lived nuclear RNA ranged in lengths up to several tens hnRNAs because about 50% of all nuclear RNA syn- of kilobases, much longer than most cytoplasmic thesis in growing mammalian cells in culture is syn- mRNAs, and was referred to as heterogeneous nuclear thesis of the single ∼13.7-kb pre-rRNA precursor. RNA (hnRNA) (3, 4).
    [Show full text]
  • Transposon Regulation Upon Dynamic Loss of DNA Methylation Marius Walter
    Transposon regulation upon dynamic loss of DNA methylation Marius Walter To cite this version: Marius Walter. Transposon regulation upon dynamic loss of DNA methylation. Development Biology. Université Pierre et Marie Curie - Paris VI, 2015. English. NNT : 2015PA066672. tel-01375673 HAL Id: tel-01375673 https://tel.archives-ouvertes.fr/tel-01375673 Submitted on 3 Oct 2016 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Université Pierre et Marie Curie Génétique et Biologie du École doctorale Complexité du Vivant developpement Institut Curie` CNRS U934 - INSERM UMR 3215 Transposon regulation upon dynamic loss of DNA methylation Thèse de Doctorat de Biologie présentée par Marius WALTER et dirigée par Déborah BOURC'HIS Présentée et soutenue le 10 Décembre 2015 devant un jury composé de: Dr. Antonin Morillon President du jury Pr. Wolf Reik Rapporteur Pr. Donal O'Carroll Rapporteur Dr. Vincent Colot Examinateur Dr. Michael Weber Examinateur Dr. Déborah Bourc'his Directrice de thèse 2 “There is large amount of evidence which suggests, but does not prove,
    [Show full text]
  • United States Patent (19) 11 Patent Number: 6,165,793 Stemmer (45) Date of Patent: *Dec
    USOO6165793A United States Patent (19) 11 Patent Number: 6,165,793 Stemmer (45) Date of Patent: *Dec. 26, 2000 54 METHODS FOR GENERATING WO 94/03596 2/1994 WIPO. POLYNUCLEOTIDES HAVING DESIRED WO 94/O9817 5/1994 WIPO. CHARACTERISTICS BY ITERATIVE WO 94/11496 5/1994 WIPO. SELECTION AND RECOMBINATION WO 94/13804 6/1994 WIPO. WO95/17413 6/1995 WIPO. WO95/22625 8/1995 WIPO. 75 Inventor: Willem P. C. Stemmer, Los Gatos, WO 96/33207 10/1996 WIPO. Calif. WO 97/07205 2/1997 WIPO. WO 97/20078 6/1997 WIPO. 73 Assignee: Maxygen, Inc., Redwood City, Calif. WO 97/25410 7/1997 WIPO. * Notice: This patent is Subject to a terminal dis WO 97/35966 10/1997 WIPO. claimer. WO 98/O1581 1/1998 WIPO. WO 98/28416 7/1998 WIPO. WO 98/41622 9/1998 WIPO. 21 Appl. No.: 09/075,511 WO 98/41623 9/1998 WIPO. 22 Filed: May 8, 1998 WO 98/41653 9/1998 WIPO. WO 98/42832 10/1998 WIPO. Related U.S. Application Data OTHER PUBLICATIONS 63 Continuation of application No. 08/621,859, Mar. 25, 1996. Atreya et al., “Construction of in-frame chimeric plant genes by simplified PCR strategies.” Plant Mol. Biol., 51 Int. Cl." ............................. C12Q 1/68; CO7H 21/02; 19:517-522 (1992). C07H 21/04; C12N 15/00 Bock et al., “Selection of single-stranded DNA molecules that bind and inhibit human thrombin,” Nature, 52 U.S. Cl. ............................. 435/.440; 435/6; 536/23.1; 355:564–566 (Feb. 2, 1992). 536/24.3; 935/76; 935/77; 935/78 Clackson et al., “Making antibody fragments using phage display libraries,” Nature, 352:624–628 (Aug.
    [Show full text]