Factors involved in 3' splice site selection in eukaryotic pre-messenger RNA.

The work presented in this thesis is an investigation of factors involved in 3' splice site selection. To try to determine the intrinsic strengths of 3' splice site sequences, a cis-competition assay system was used. This system was based on the large intervening sequence (IVS-2) of the rabbit beta-globin gene. Synthetic 3' splice site sequence oligonucleotides were inserted into the EcoRl restriction site, forty nine nucleotides downstream of the authentic rabbit beta-globin IVS-2 3' splice site. The oligonucleotides conformed to the established 3' splice site consensus sequence and allowed for variations within this sequence. The authentic site served as a constant reference site against which the strengths of the synthetic sites could be measured. When spliced in HeLa cells in vivo, all constructs tested were seen to choose the authentic 3' splice site over the synthetic 3' splice site under test. A series of mutageneses was carried out to try to decrease the intrinsic strength of the authentic site and/or improve the environment of the synthetic site such that the overall strengths of the two sites might be balanced. An AG→CG mutation at the authentic 3' splice site caused the synthetic 3' splice site to be activated as a cryptic site in vivo and in vitro. In this case lariat formation was mapped to an artificially created branch point within exon 3. Splicing component binding to both 3' splice sites was investigated by looking at protection of the RNA from oligonucleotide directed cleavage by RNase H. Initial protection of both 3' splice sites was independent of the final choice of site. However, branch point protection was dependent on the 3' splice site chosen. Components bound to the authentic 3' splice site could be immunoprecipitated whether that site was chosen or not. The synthetic 3' splice site was poorly precipitated even when it was chosen. This data tends to suggest that the synthetic 3' splice site directs inefficient complex assembly, and that at least partial complex assembly occurs at a 3' splice site which has been inactivated by an AG→CG mutation. Preliminary work was carried out to develop a method for the analysis of splicing component binding to either or both 3' splice sites of material within fully and partially assembled splicing complexes (spliceosomes) isolated by sucrose gradient sedimentation.