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Title: RNA interference and heterochromatin formation in fission yeast
Author: Almeida, Ricardo
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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There is strong evidence suggesting that RNAi acts co-transcriptionally in order to promote heterochromatin formation. Thus, it is possible that RITS activity is interlinked with transcription-related processes such as cleavage/poly-adenylation, transcription termination and RNA turnover by the exosome complex. In order to investigate this hypothesis, the integrity of RNAi and heterochromatin was assayed in mutants for factors that are involved in all three pathways. Mutations on dhp1 (termination), pfs2 (cleavage and polyadenylation) dis3 and rrp6 (exosome) had negligible effects on RNAi activity and heterochromatin-mediated silencing with only the exosome showing some involvement in the downstream degradation of centromeric transcripts. Conventional RNAi enforces post-transcriptional repression by targeting mRNA molecules for degradation. This is mediated by the endonuclease activity of Argonaute (“slicing”). Although the key residues for this activity are conserved between human Ago2 and S. pombe Ago1, the importance of this “slicing” activity to heterochromatin assembly was not clear. Mutations were made in putative catalytic residues on the endogenous ago1 gene in order to address this question. These mutations severely affect the activity of RNAi in fission yeast and destabilize the heterochromatin structure at centromeres. Consequently, centromere function is affected and chromosome segregation is deficient. Ago1 localization to the centromeres is impaired in these mutants and cannot nucleate heterochromatin nucleation though siRNA production is not fully abolished. Thus, Ago1 slicing activity is crucial for sustainable RNAi and its role in heterochromatin integrity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available