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Title: Functional analysis of Prp45p, a pre-mRNA splicing factor in Saccharomyces cerevisiae
Author: Muraru, Mariela I.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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In this study we aimed to understand the role Prp45p executes within the spliceosome and the splicing mechanism. Part of this work was a detailed investigation into the relationships between the structure and the function of this protein by employing a screen for temperature-sensitive mutants. Prp45p is known to associate with the spliceosome throughout the splicing reactions but at what stage the protein is involved in spliceosome assembly i.e. pre-spliceosome, inactive or active spliceosome remained to be determined. Using a PRP45 conditionally regulated strain, it was found upon depletion of Prp45p from yeast cells and analysis of the in vivo splicing systems by native gel fractionation that the formation of the active spliceosome does not take place. This behaviour is compatible with Prp45p being a component of the Ntc-protein complex, which is known to be involved in this step of spliceosome assembly. Moreover, co-immunoprecipitation experiments with a tagged allele of the splicing factor Prp46p, confirmed the interaction between these two proteins suggested by two-hybrid screens. Using random PCR mutagenesis, there were identified two mutants with growth defects at 37°C. The mutants, named prp45-57 and prp45-113 contained mutations in two regions, designated A and B and located respectively upstream and, downstream of the absolutely conserved SNWKN motif. To understand which of the mutations were responsible for the temperature-sensitive phenotype, the substitutions in region A and/or region B were recreated by site-directed mutagenesis. It was proved that the phenotype requires mutations in both segments, which strongly suggests that regions A and B together play roles in protein function, perhaps through intra- or inter-protein interactions. However, co-immunoprecipitation experiments revealed that these substitutions in Prp45p do not alter the interaction with Prp46p in vitro. In order to investigate the role of these mutations within the cell, two strains were created that carry these mutations in a c-Myc tagged PRP45 ORF. When growing these strains at non-permissive temperature and employing a β-galactosidase assay, it was found that they had a mild effect on pre-mRNA splicing and do not affect the transcription/translation of the reporter genes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available