Molecular characterisation of Saccharomyces cerevisiae Prp17protein and its interacting partners

Abstract

In majority of eukaryotic genes, removal of intervening sequences from pre?mRNA is essential for its expression. A ribonucleoprotein complex called the spliceosome catalyses the removal of intron by two sequential phosphotransesterification reactions. Analyses of spliceosomal components and their roles in the splicing reaction have been studied using Saccharomyces cerevisiae as a model system. Prp17p is one of the components of the spliceosome that is required specifically for the second step of the splicing reaction both in vivo and in vitro. The Prp17 protein contains four copies of the conserved G?beta or WD40 repeat units. This conserved protein motif is found in diverse proteins and its hypothesised function is to mediate protein–protein interactions. In a recent study, an allele of prp17 (cdc40?1) was defined as a gene required for the S?phase progression and mitotic spindle assembly/maintenance during the cell cycle. Thus Prp17p may provide a link between two important biochemical pathways: splicing and cell cycle, in yeast. Domain architecture of Prp17 protein For a better understanding of the functional domains in Prp17 protein that mediate its two variant roles, a PCR?based mutagenesis screen is underway. In this strategy the cloned full?length PRP17 Open Reading Frame will be subjected to PCR amplification under conditions of reduced fidelity for the polymerase. This will generate a random pool of mutations which will be screened in vivo by a gap?repair strategy for mutations in the plasmid?borne copy of PRP17 that do not complement the chromosomal prp17 mutations. Plasmids that confer such phenotypes will be sequenced to determine the nature and position of the mutations in PRP17. So far various prp17 mutant strains that will be used in these studies have been characterised genotypically. These strains will be used for transformation experiments. New mutants of prp17 that will be generated will be analysed for defects in splicing and cell?cycle progression. Prp17 interacting proteins Previous studies in our lab have identified spontaneous extragenic suppressors of prp17 mutants and also used the yeast two?hybrid assay to define interacting partners for Prp17 and that led to the identification of two new factors Pip1 and Pip4 (Prp?interacting proteins). Both of these proteins are unique to the database with no obvious homologs and/or conserved motifs. I have initiated studies involving functional characterisation of Pip1 to decipher its cellular role. Cloning of two overlapping genomic fragments of PIP1 by PCR amplification is initiated and the 3' fragment of PIP1 was cloned into PCR?script plasmid, cloning of the 5' fragment of PIP1 is in progress. The cloned products will be used to generate overexpressed fusion proteins in E. coli and in turn will be used to raise polyclonal antisera. Meanwhile full?length PIP1 ORF was cloned in our lab, which was subcloned into pRSETA for overexpression of full?length PIP1. The antisera will be used for in vivo immunolocalisation of Pip1 and also to study the effects of immunodepletion of the protein on in vitro splicing extract. These studies will help in elucidating the cellular function of Pip1 protein.