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Ciona Intestinalis: Molecular Characterization of the SL RNA Spliced leader (SL) trans-splicing in the ascidian tunicate Ciona intestinalis: molecular characterization of the SL RNA Brendan Yeats Biology Department McGill University, Montreal August 2009 A thesis submitted to McGill University in partial fulfilment of the requirements of the degree of MSc, biology. © Brendan Yeats 2009 Library and Archives Bibliothèque et Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l’édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre référence ISBN: 978-0-494-61656-7 Our file Notre référence ISBN: 978-0-494-61656-7 NOTICE: AVIS: The author has granted a non- L’auteur a accordé une licence non exclusive exclusive license allowing Library and permettant à la Bibliothèque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l’Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans le loan, distribute and sell theses monde, à des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, électronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L’auteur conserve la propriété du droit d’auteur ownership and moral rights in this et des droits moraux qui protège cette thèse. Ni thesis. Neither the thesis nor la thèse ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent être imprimés ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author’s permission. In compliance with the Canadian Conformément à la loi canadienne sur la Privacy Act some supporting forms protection de la vie privée, quelques may have been removed from this formulaires secondaires ont été enlevés de thesis. cette thèse. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n’y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. ABSTRACT I initially set out to identify the cap structure on the spliced leader RNA of Ciona intestinalis. During this investigation, I discovered a previously unobserved 53 nt transcript containing the spliced leader sequence. This transcript contained the usual metazoan spliced leader RNA cap, trimethylguanosine, while the canonical spliced leader RNA lacked this moiety, but likely contained a m7G cap. The cap structure of the trans-spliced troponin I mRNA matched that of the canonical spliced leader RNA, indicating that the canonical spliced leader RNA, not the 53 nt transcript, is the donor for troponin I. Further immunoprecipitation studies showed that both the canonical spliced leader RNA and the novel 53 nt transcript exist in association with Sm proteins. I cloned a genomic DNA segment containing four tandem repeats of the spliced leader RNA gene, and this was used as a probe in an in situ hybridization study that showed the vast majority of the spliced leader RNA genes reside on chromosome 8. Finally, I performed some preliminary work showing that outrons, the 5‟- segments of pre-mRNAs removed by trans-splicing, may exist in sufficient quantities as to be detected by PCR amplification. ABSTRAIT Au d‟épart, j'ai entrepris d‟identifier la structure coiffe de l‟ARN spliced leader de Ciona intestinalis. Durant cette recherche, j'ai découvert un nouveau transcript d‟RN de 53 nt qui contient la séquence spliced leader. Ce transcrit contient la coiffe usuele des ARNs spliced leader des métazoaires, le trimethylguanosine, tandis que l‟ARN spliced leader canonique ne possède pas cette modification, mais contient, fort probablement, une coiffe m7G. La structure de la coiffe de l‟ARNm de la troponine I trans-épissé correspond à celle de l‟ARN spliced leader canonique, indiquant que l‟ARN spliced leader est le donneur pour la troponine I, et non l‟ARN de 53 nt. Des études d'immunoprécipitation supplémentaires ont montré que l‟ARN spliced leader canonique et le nouvel ARN de 53 nt existent en association avec des protéines Sm. J'ai cloné une sequence d‟ADN génomique contenant quatre repetitions en tandem du gène l‟ARN spliced leader. Ce clone a été utilisé comme sonde lors d‟une experience d‟hybridation in situ qui a montré que la grande majorité des gènes l‟ARN spliced leader réside sur le chromosome 8. Finalement, j'ai effectué une étude préliminaire montrant que les outrons, les segments 5' des ARNs pré-messagers enlevés par le trans-épissage, existent en quantité suffisante et peuvent être détecté par l'amplification PCR. TABLE OF CONTENTS 1. Introduction Overview of the thesis............................................................................................ 1 Background ............................................................................................................ 2 Spliced leader trans-splicing......................................................................... 2 Phylogenetic distribution of SL trans-splicing ............................................. 4 Characteristics of SL RNAs .......................................................................... 6 Functions of SL trans-splicing ...................................................................... 9 Synthesis of the TMG cap precursor m7G ................................................. 14 Synthesis and function of TMG caps .......................................................... 14 Sm proteins, snRNPs, and splicing ............................................................. 19 Ciona intestinalis ........................................................................................ 21 Project rationale ................................................................................................... 22 Molecular characterization of the Ciona intestinalis SL RNA ................... 22 FISH to the Ciona intestinalis SL RNA gene cluster.................................. 23 Outron amplification ................................................................................... 23 2. Methods RNA extraction .................................................................................................... 26 Northern blotting.................................................................................................. 26 PCR amplification and cloning of the 53 nt SL RNA exon-containing transcript............................................................................................................... 28 Reverse transcription............................................................................................ 29 Surveying the expression of various SL RNAs ................................................... 29 Immunoprecipitation using the K121 mouse monoclonal antibody .................... 30 Immunoprecipitation using the R1131 rabbit polyclonal antiserum.................... 30 Shrimp alkaline phosphatase (SAP) treatment .................................................... 31 Tobacco acid pyrophosphatase (TAP) treatment ................................................. 31 Northern blotting to determine of the cap status of the SL RNA ........................ 32 Immunoprecipitation using the H20 mouse monoclonal antibody ...................... 33 Immunoprecipitation using the anti-Sm antiserum .............................................. 33 Identification of Sm-Associated transcripts ......................................................... 34 RNase H digestion of Ciona intestinalis TnI mRNA ........................................... 34 Measuring hybridization on Northern blots ......................................................... 35 Topo TA® cloning ............................................................................................... 36 Production of a fluorescent in situ hybridization probe ....................................... 37 Fluorescent in situ hybridization.......................................................................... 39 Amplification of the TnI outron........................................................................... 39 3. Results: characterization of the Ciona intestinalis SL RNA in terms of size heterogeneity, cap structure, association with Sm proteins, and expression profile Size heterogeneity amongst SL-related transcripts .............................................. 42 The 53 nt SL RNA exon-containing transcript is generated by trans-splicing .... 45 Survey of SL RNA size and sequence variation .................................................. 47 K121 immunoprecipitation indicates the Ciona intestinalis SL RNA is most likely not TMG-capped, while the 53 nt SL RNA exon-containing transcript is 49 R1131 immunoprecipitation confirms the Ciona intestinalis SL RNA is not TMG-capped, while the 53 nt SL RNA exon-containing transcript is ................ 54 The 5‟-end of the SL RNA is phosphatase protected and therefore capped ........ 58 The Ciona intestinalis SL RNA cap is most likely m7G ..................................... 64 The Ciona intestinalis SL RNA, as well as the 53 nt SL RNA exon-containing transcript, are snRNPs associated with Sm proteins ............................................ 66 The canonical ~46 nt SL RNA, not the 53 nt SL RNA exon-containing transcript, is the SL donor for TnI........................................................................................
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