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 <i>PRP45</i> conditionally regulated strain, it was found upon depletion of Prp45p from yeast cells and analysis of the <i>in vivo</i> 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 <i>prp45-57 </i>and <i>prp45-113</i> contained mutations in two regions, designated <i>A</i> and <i>B</i> 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 <i>A</i> and/or region <i>B </i>were recreated by site-directed mutagenesis. It was proved that the phenotype requires mutations in both segments, which strongly suggests that regions <i>A </i>and <i>B </i>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 <i>in vitro</i>. 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 <i>PRP45</i> 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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:659761 |
| Date | January 2003 |
| Creators | Muraru, Mariela I. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/15461 |
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