The long term goal of the proposed research is to understand the role of SR proteins in spliced leader (SL) addition trans splicing int he nematode Ascaris lumbricoides. This is a reaction that adds a short exon, the SL to the 5' end of pre-mRNA transcripts. Recent developments in the field of pre-mRNA cis splicing in higher eukaryotes have demonstrated that SR protein splicing factors play an important part in many different aspects of constitutive and regulated splicing. An in vitro system from A. lumbricoides embryos has been described that catalyzes both trans and cis splicing. The experiments proposed here will examine the involvement of SR proteins in trans splicing, and with a particular emphasis on comparisons to their roles in cis-splicing reactions i both A. lumbricoides and in mammalian tissue culture cells.
Five specific aims are proposed; 1. To isolate individual A. lumbricoides SR proteins and to clone their genes. 2. To examine the effects of A. lumbricoides SR protein on trans and cis - splicing in vitro and to compare these results to mammalian cis-splicing reactions. 3. To examine in detail the interaction of the A. lumbricoides SR proteins with the SL RNA/RNP. 4. To determine the protein-protein interactions among A. lumbricoides SR proteins as well as among the SR proteins and other cellular factors. 5. To determine the difference in the expression/post-transcriptional modification of the A. lumbricoides SR proteins throughout development. The experiments proposed in this application will allow the role of SR proteins to be determined in trans splicing and therefore represent a new direction in the examination of this RNA processing reaction that is so widespread among lower eukaryotes. Many of these organisms are medically important and a complete dissection of the trans-splicing reaction may provide a route to the generation of novel therapeutics.
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