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Title: Characterisation of factors affecting RNAi-directed heterochromatin assembly in fission yeast
Author: White, Sharon Ann
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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In fission yeast, heterochromatin assembly requires an intact RNA interference (RNAi) pathway. Non-coding RNA transcripts originating from centromeric repeats are found to be associated with protein complexes that mediate RNAi. These transcripts are processed and the resulting siRNAs direct heterochromatin assembly over the homologous repeats and induce transcriptional silencing. The centromere:suppressor of position effect (csp) mutants were previously isolated as specifically alleviating silencing within the centromeric outer repeats. The initial aim was to identify and characterise additional factors by analysing several mutations in unknown genes and then investigate their role in centromeric heterochromatin formation and integrity. Complementation with genomic libraries followed by sequencing allowed the identification of csp7, csp9, csp10 and csp12 as alleles of rdp1+, ago1+, cid12+and arb1+ respectively. cid12, which encodes a putative poly(A) polymerase and associates in a complex with Rdp1, was chosen for more detailed analyses. Mutations in the predicted catalytic domain of Cid12 which would be expected to abrogate its function were generated (Cid12dada). Recombinant wild type and mutant protein were produced and employed in in vitro assays designed to test the biochemical function of Cid12. To date no convincing poly(A) polymerase activity has been detected however, assays indicate that Cid12 may possess nuclease activity. Affinity selection indicates that Cid12 associates with many proteins. Such interactions may be constitutive or transient. It is possible that Cid12 is only active in the context of these proteins and/or that it has unusual, unknown requirements with respect to substrate specificity. These analyses are described in detail and demonstrate that Cid12 plays a central role in heterochromatin assembly at centrosomes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available