: The broad long term goal of the proposed research is to understand in detail the mechanism of nuclear pre-mRNA splicing in parasitic nematodes using Ascaris as a model system. Cell free extracts prepared from developing embryos of this organism efficiently catalyze both cis and trans-splicing; these extracts remain the only experimental system in which it is possible to analyze trans-splicing using biochemical approaches. Recent studies have identified and characterized two protein factors, SL-30 and SL-95 that are uniquely required for trans-splicing. These proteins function to promote joining of the 5' and 3' splice sites. Recent studies have also revealed that c/s-splicing in nematodes results in the deposition of an exon junction complexon mature mRNAs; to date this complex has only been analyzed in mammalian cells. These observations serve as the basis for future mechanistic analyses, which will first use a depletion/reconstitution approach to study further the function of the trans-splicing specific factors. In addition, fusion protein strategies will be employed to determine if the SL-30 and SL-95 are not only necessary but sufficient for SL RNP function. Second, the evidence indicates that SL-30 makes essential contacts with the splicing factor SF1/BBP, a protein that plays a bridging role between 5' and 3' splice sites in cis-splicing. Depletion/reconstitution will be used to determine which domains of SF1/BBP are required for both cis and trans-splicing. In addition, a sensitive competition assay will be used to determine which region of SF1/BBP communicates with factors at cis 5' splice sites. Finally, the availability of homologous Ascaris extracts that catalyze both splicing and efficient in vitro translation provides a unique opportunity to biochemically address the function of the EJC. Sensitive reporter mRNAs will be used to determine if the EJC enhances translation and, if enhancement is observed, experiments will be conducted to determine the mechanism by which enhancement occurs. In combination, the proposed studies should provide new insight into the mechanism of trans-splicing in particular and splicing in general. They may also provide novel information relevant to the coupling of splicing and post-processing mRNA metabolism.
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