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Regulaɵon of the Minor Spliceosome Through Alternaɵve Splicing And

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Regulaɵon of the Minor Spliceosome Through Alternaɵve Splicing And JENS VERBEEREN RegulaƟ on of the Minor Spliceosome through AlternaƟ ve Splicing and Nuclear RetenƟ on of the U11/U12ͳ65K mRNA Institute of Biotechnology and Division of Genetics Department of Biosciences Faculty of Biological and Environmental Sciences and Doctoral Programme in Integrative Life Science University of Helsinki ACADEMIC DISSERTATION To be presented for public examination with the permission of the Faculty of Biological and Environmental Sciences in Auditorium 1041 in Biocenter 2 (Viikinkaari 5, Helsinki) on January 29th 2016, at 12 noon. Helsinki 2016 Custos Professor Juha Partanen Department of Biosciences University of Helsinki Helsinki, Finland Supervisor Docent Mikko Frilander Institute of Biotechnology University of Helsinki Helsinki, Finland Th esis advisory committee Professor Yrjö Helariutta Docent Petri Auvinen Sainsbury Laboratory Institute of Biotechnology University of Cambridge University of Helsinki Cambridge, United Kingdom Helsinki, Finland Reviewers Docent Tapio Heino Docent Noora Kotaja Department of Biosciences Department of Physiology University of Helsinki Institute of Biomedicine Helsinki, Finland University of Turku Turku, Finland Opponent Associate Professor Stephen M. Mount Department of Cell Biology and Molecular Genetics University of Maryland College Park, Maryland, United States Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis 8/2016 ISBN 978-951-51-1856-1 (paperback) ISBN 978-951-51-1857-8 (PDF) ISSN 2342-3161 (Print) ISSN 2342-317X (Online) Layout: Tinde Päivärinta/PSWFolders Oy Hansaprint Helsinki 2016 http://ethesis.helsinki.fi “Die sijn tijtjen weet te gissen, En sijn toutjen weet te splissen, En sijn glas te roer te staen, Mag wel voor een bootsman gaen” Jacob Cats, 1632 TABLE OF CONTENTS List of original Publications ..................................................................................................... vi Abbreviations ...................................................................................................................... vii Abstract ..................................................................................................................... viii 1. Review of the Literature ......................................................................................................1 1.1 Introns ........................................................................................................................ 1 1.1.1 Defi nition and Classifi cation .....................................................................................1 1.1.1.1 Spliceosomal Introns ....................................................................................1 1.1.1.2 tRNA Introns .................................................................................................4 1.1.1.3 Group I Introns .............................................................................................4 1.1.1.4 Group II Introns ...........................................................................................5 1.1.2 On the Origin of Introns ............................................................................................6 1.1.2.1 Intron Gain and Intron Loss .......................................................................7 1.1.2.2 Roots of the U12-type Intron .......................................................................8 1.1.3 What good is an Intron? ............................................................................................8 1.2 RNA Splicing and the Spliceosome ........................................................................ 9 1.2.1 Spliceosome Composition ...................................................................................... 10 1.2.2 Exon and Intron Defi nition ....................................................................................12 1.2.2.1 Splicing Enhancers and Splicing Silencers ............................................. 13 1.2.2.2 Splicing Activators and Repressors ......................................................... 13 1.2.3 Spliceosome Assembly and Catalysis .................................................................... 15 1.2.3.1 Major Spliceosome Assembly .................................................................. 15 1.2.3.2 Minor Spliceosome Assembly ..................................................................17 1.2.4 Alternative Splicing.................................................................................................. 18 1.2.4.1 Regulatory Role of Alternative Splicing ................................................ 19 1.2.4.2 Alternative Splicing of U12-type Introns ............................................... 19 1.3. Splicing and other pre-mRNA Processes ..................................................................20 1.3.1 Th e Timing of Splicing ............................................................................................ 20 1.3.2 Splicing and Nuclear Export ...................................................................................21 1.3.3 Splicing and Quality Control: Nonsense Mediated Decay ................................. 22 1.3.4 Splicing and 3´ End Processing ............................................................................. 22 1.4 Th e Minor Spliceosome: Signifi cance .......................................................................23 1.4.1 Function and Regulation of the Minor Spliceosome .......................................... 23 1.4.2 Minor Spliceosome and Disease ............................................................................ 25 2. Aims of the Study ...............................................................................................................26 3. Materials and Methods ......................................................................................................27 4. Results and Discussion ......................................................................................................28 4.1 Ultraconserved non-coding regions in minor spliceosome-associate genes are linked to alternative splicing ....................................................................28 4.2 U11 binds the conserved enhancer and activates alternative splicing through exon defi nition interactions mediated by the U11-35K protein................30 4.3 Alternative splicing leads to a multilayered inhibitory mechanism for 48K and 65K mRNA expression ...............................................................................31 4.4 USSE provides negative auto-regulation for the 48K gene and minor spliceosome mediated cross-regulation for the 65K gene ........................................33 4.5 Evolutionary role of the USSE ...................................................................................34 5. Concluding Remarks .........................................................................................................37 6. Acknowledgements ............................................................................................................38 7. References ...........................................................................................................................40 LIST OF ORIGINAL PUBLICATIONS Th e thesis is based on the following articles, referred to in the text by their Roman numerals: I. Verbeeren J*, Niemelä EH*, Turunen JJ*, Will CL, Ravantti JJ, Lührmann R, Frilander MJ. An ancient mechanism for splicing control: U11 snRNP as an activator of alternative splicing. Mol Cell 2010; 37(6):821-33. (Reproduced here with permission from Elsevier) II. Niemelä EH, Verbeeren J, Singha P, Nurmi V, Frilander MJ. Evolutionarily conserved exon defi nition interactions with U11 snRNP mediate alternative splicing regulation on U11- 48K and U11/U12-65K genes. RNA Biol 2015; 12(11):1256-64. (Reproduced here with permission from Taylor & Francis) III. Verbeeren J, Niemelä EH, Frilander MJ. Regulation of the minor spliceosome U11/U12- 65K mRNA by nuclear retention and transcriptional read-through. Unpublished manu- script. * Equal contribution Th e author´s contribution to each article: I. JV planned experiments and analyzed alternative splicing of the U11/U12-65K gene in the contexts of mutations of the regulatory element, knock-down of minor spliceosomal pro- teins, inhibition of nonsense mediated decay, and morpholino transfections. JV planned and performed the critical genetic rescue experiments, performed the luciferase assays, and wrote the article together with the other authors. II. JV designed experiments related to the investigation of USSE distance requirements and supervised one undergraduate student who performed them. JV supervised another un- dergraduate student who performed the initial U11-35K tethering experiments. JV con- ducted experiments to evaluate the role of the 35K and other proteins in splicing activation (RT-PCR splicing analysis and qPCR), and wrote the article together with EN and MJF. III. JV planned and performed virtually all experiments (single molecule FISH, qPCR, cellular fractionation, RNase protection assay, and RT-PCR splicing analysis), and wrote the article together with MJF. vi ABBREVIATIONS A adenine BP branch point BPS branch point sequence C cytosine CP cleavage/polyadenylation EJC exon junction complex ESE exonic splicing enhancer ESS exonic splicing silencer EST expressed sequence tag FISH fl uorescence in situ hybridization G guanine hnRNP heterogeneous nuclear ribonucleic protein IEP intron-encoded protein ISE intronic
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