Uncovering Mechanisms of 5' Splice Site Fidelity

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PROJECT SUMMARY/ABSTRACT Precursor messenger RNA (pre-mRNA) splicing is essential in higher eukaryotes in order to produce functional messenger RNAs to code for proteins. The splicing reaction is carried out by a large macromolecular machine called the spliceosome. The spliceosome assembles onto pre-mRNA substrates through a complex binding, rearrangement and release of 5 small nuclear RNAs and over 100 associated proteins. The exact roles of many of the spliceosome-associated factors are poorly understood. Our lab uses the microscopic worm C. elegans as a model to explore pre-mRNA splicing through integrated genetic, genomic and biochemical approaches. We have established sensitives genetic screens to identify factors important for the accurate assembly of the spliceosome at the 5' splice site. Our data suggest the hypothesis that key spliceosomal components have an important role in securing the accurate transfer of the 5' splice site from its initial recognition by U1snRNP to its loading into the active site of the spliceosome. The dominant suppressor mutations that we uncovered fall into two classes; 1) those that allow the spliceosome to remain in an open conformation, which allows the 5' splice site position to slide between two regions 23nt apart during loading into the active site and 2) those that promote the usage of an intron that begins with an unusual UU dinucleotide. We will explore the following questions: 1. How do protein factors control the accurate selection of 5' splice sites during spliceosome assembly? We hypothesize that components of the assembled spliceosome control the final determination of 5' splice site selection. We will study how alleles of PRPF8, SNRNP27 and SNRNP200 (Brr2) affect precise 5' splice site usage in worms. Because of the prevalence of human genetic disease alleles with mutations identical to the ones explored in our genetic screen, we also plan to take our studies into human cells to determine if the alleles identified in worms can be targets for translational research into splicing regulation. 2. How do KIN17 and PRCC maintain 5'ss fidelity? We have found a phenomenon in which two 5'ss separated by a single nucleotide are both used even in the presence of a wild type spliceosome. We have identified a class of dominant suppressors that promote usage of introns that begin with UU instead of the canonical GU dinucleotide. These alleles are in the worm homologs of human KIN17 and PRCC; little is known about a role for either protein in splicing other than that they transiently interact with mammalian Bact spliceosomes. We now have functional splicing assays for these factors to exploit in characterizing their function in maintaining splice site fidelity.