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Title: Analysis of yeast PRP8 protein and its role in pre-mRNA splicing
Author: Brown, Jeremy David
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1992
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PRP8 protein is a component of the nuclear pre-mRNA splicing machinery. The PRP8 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 in vivo and heat-inactivation of temperature-sensitive forms of the protein. The in vivo 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 in vitro 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 prp8 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 PRP8 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) In vivo 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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available