The Prp2 protein of <I>Saccharomyces cerevisiae </I>is a 100kDa non-snRNP splicing factor with amino acids common to the DEAD/H-box family of ATP-dependant RNA helicases. Prp2, Prp16 and Prp22 proteins have extensive sequence similarity, and function at sequential steps in the splicing pathway. Prp2 and Prp16 form transient reactions with the spliceosome, Prp2 at step 1 and Prp16 at step 2. In addition to the conserved motifs of the DEAD/H-box family, Prp2 also contains a sequence resembling a zinc finger motif. This sequence has been extensively mutagenised, and two mutations were found by <I>in vivo </I>complementation assays to affect the function of Prp2. In order to study the molecular interactions of Prp2 at step 1, several dominant negative <I>PRP2</I> alleles had been generated previously in this laboratory by site-directed mutagenesis. When overexpressed from an inducible promoter, these cause accumulation of pre-mRNA in the presence of wild-type <I>PRP2. </I>These dominant negative alleles and wild-type <I>PRP2 </I>were subjected to deletion analyses. In the case of one dominant negative mutant which has a GKT-AKT change in the ATP-binding motif, some dominant activity was retained despite the removal of 19kDa from the protein. It has been suggested that the Prp16 protein plays a proof-reading role in splicing, as a mutant of <I>PRP16, prp16-1,</I> suppresses an A-C change of the branch nucleotide. A mutation analogous to the <I>prp16-1</I> allele was created in <I>PRP2 </I>to determine if Prp2 plays a role in influencing the fidelity of splicing. Although no effect was found for this and other mutants of <I>PRP2, </I>it was discovered that overexpression of wild-type <I>PRP16</I> had an effect similar to that of the <I>prp16-1 </I>mutant on the fidelity of splicing. A new hypothesis is proposed for the mechanism of suppression of branchpoint mutants by Prp16 whereby the Prp16 protein might mediate the binding of an unknown proof-reading factor to the branchpoint.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:640492 |
| Date | January 1996 |
| Creators | Anderson, Susan Isobel |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13396 |
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