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Title: Identification and characterisation of Prp45p and Prp46p : two novel pre-mRNA splicing factors in Saccharomyces cerevisiae
Author: Albers, Michael
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1999
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We set out to investigate protein-protein interactions within the spliceosome of the budding yeast Saccharomyces cerevisiae by performing exhaustive two-hybrid screens using well characterised splicing proteins as baits. This approach should allow the identification of i) novel splicing proteins, ii) novel interactions between known splicing factors and iii) links between splicing and other cellular pathways, especially processes of mRNA metabolism. Prp22p is an RNA-helicase with at least two distinct functions in the splicing pathway. It is required for the second transesterification reaction to proceed and in addition it has a role during spliceosome disassembly. When Prp22p was used as bait in a two-hybrid screen, the most statistically interacting protein found was the Fun20 protein. The 42 kDa Fun20 protein was previously shown to be essential for cell viability, but not further characterisation had been performed (Fun = function unknown). In order to investigate whether the protein plays a role in pre-mRNA splicing, a strain was generated carrying a protein A-tagged and conditionally regulated FUN20 gene. Growing the strain under non-permissive conditions leads to an accumulation of pre-mRNA within the cell, showing that the Fun20 protein is indeed required for splicing in vivo. The protein was renamed Prp45p to indicate its role in pre-mRNA processing. Using the epitope-tagged versions of the protein in co-immunoprecipitation experiments, it was found that Prp45p co-precipitates pre-mRNA, splicing reaction-intermediates, the spliced exons and the excised intron from cell extracts. This strongly suggests that Prp45p is associated with the spliceosome throughout the splicing reactions. Furthermore the tagged-protein weakly co-precipitates the U2, U5 and U6 snRNAs from cell extracts, showing its association with a subset of spliceosomal snRNPs. Depletion of Prp45p from cell extracts completely abolishes splicing of added actin pre-mRNA in these extracts. However, splicing activity can be at least partially restored by adding back recombinant HIS6-tagged Prp4p, that had been produced in Escherichia coli and affinity purified. This shows that Prp4p is required for splicing in vitro and furthermore suggests a role for the protein before the first transesterification takes place. Prp45p was then used as bait in a two-hybrid screen and interacted strongly with a protein of unknown function encoded by ORF YPL151c.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available