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 mP7PG 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. / 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 mP7PG. 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.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.67011 |
| Date | January 2009 |
| Creators | Yeats, Brendan |
| Contributors | Kenneth E M Hastings (Supervisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | French |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Biology) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | Electronically-submitted theses. |
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