| Summary: | PRP8 protein is a component of the nuclear pre-mRNA splicing machinery. The <i>PRP8</i> gene had previously been cloned from yeast and antibodies raised against the protein. Using these it had been shown that PRP8 protein is a component of the U5 snRNP, U4/U6.U5 tri-snRNPs and of the spliceosome, in which it contacts the substrate RNA. In this thesis I present further work on the characterisation of the roles of the PRP8 protein in splicing complex assembly and splicing using these antibodies and two other approaches: genetic depletion of the protein <i>in vivo</i> and heat-inactivation of temperature-sensitive forms of the protein. The <i>in vivo</i> depletion of an intrinsic snRNP protein has not previously been reported and this approach allowed a more definitive investigation of the function of PRP8 protein.(i) Antibodies which recognise the native PRP8 protein inhibited splicing <i>in vitro</i> and this was shown to be due to a block in spliceosome assembly. A pre-spliceosome complex containing the U1 and U2 snRNPs accumulated in splicing reactions inhibited by these antibodies. Anti-PRP8 antibodies also detected a change in the interactions of PRP8 protein in the spliceosome as the active complex formed.(ii) A yeast strain conditionally producing PRP8 protein was generated, and several temperature-sensitive <i>prp8</i> mutations were outcrossed from mutagenised backgrounds and the mutations mapped within the genes. Extracts in which PRP8 protein was either depleted or heat-inactivated were made from these strains and shown to be inactive for splicing. Splicing reactions carried out with these extracts accumulated a pre-spliceosome complex similar to that seen when PRP8 function was blocked by antibodies.(iii) Depletion and heat-inactivation of PRP8 protein were shown to result in loss of U4/U6.U5 tri-snRNPs, consistent with a requirement for PRP8 activity for the stable formation of tri-snRNPs, without which spliceosomes fail to form. As a consequence of PRP8 depletion the levels of U4, U5 and U6 snRNAs declined dramatically. From this result, and known genetic interactions between <i>PRP8</i> and several putative RNA helicase genes, it was proposed that without PRP8 activity aberrant tri-snRNPs form, on or in which helicase activities act to unwind RNA structures, thereby exposing the snRNAs to the action of nucleases.(iv) <i>In vivo</i> depletion of U5 snRNA was found to have little effect on either PRP8 protein or the U4 and U6 snRNAs; in fact, the levels of U4 and U6 snRNAs rose as U5 became depleted. This indicated that the loss of U4 and U6 snRNAs seen on depletion of PRP8 protein was not a consequence of loss of U5 snRNAs and favoured the model proposed in (iii). These results together with previously published data suggest that PRP8 protein activity is required for : 1) the formation of complete and functional U5 snRNPs; 2) the formation of stable tri-snRNPs; 3) interaction of tri-snRNPs with the pre-spliceosome; 4) the regulation of helicase activities associated with the tri-snRNP and the spliceosome.
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