Analysis of yeast Prp8 protein and its role in U5 snRNP assembly

• Main Author: Boon, Kum Loong
• Published: University of Edinburgh 2005
• Subjects: 579
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.641781

PRP8 protein is a component of the nuclear pre-mRNA splicing machinery. The Prp8p had previously been identified as a component of the U5 snRNP, U4/U6, U5 tri-snRNPs of the spliceosome, which contacts the catalytic centre of the spliceosome, as detected by photochemical crosslinks of Prp8p with functional important region of the U5 and U6 snRNAs, and with the 5’ splice site, 3’ splice site, and branchpoint of pre-mRNA. In this thesis, I present further work on protein interactions between Prp8p and other splicing factors using Prp8p partitioned strains. The Prp8p partitioned constructs allowed investigation of factors that associate with the N- or C-terminal regions of Prp8p by isolation of the sub-complexes associated with different Prp8 protein fragments. Thus, the U5 snRNP protein Snu114p associates with Prp8p in the region 437-770 whereas fragmenting Prp8p at residue 2173 destabilises its association with Aar2p. The role of Prp8p on U5 snRNP assembly was investigated. Upon the identification of nuclear localisation signal of Prp8p, I further investigated the role of Prp8p in U5 snRNP assembly, and showed that Prp8p-Snu114p-Aar2p-U5 complex formation happened in cytoplasm, and Prp8p-Snu114p-Brr2p-U5 association of the complex took place in the nucleus. Despite the fact that Prp8p is nuclear localised, the association between Prp8p and Brr2p is disrupted by Prp8p C-terminal mutations that resemble human Prp8p mutations in result of retinitis pigmentosa type 13. In addition, I also identified the importin, Kap95p, is needed for Snu114p, and snRNAs nuclear import. In this thesis I also characterised a Prp8p co-purified protein, Spp382p, and results indicate that Sp382p is needed in the late-stage of splicing.