The long-term goal of the proposed research is to understand in detail the molecular mechanism of spliced leader (SL) addition trans-splicing in nematodes. In this RNA processing reaction, the 5' terminal exon (SL) is donated by a trans-splicing specific Sm snRNP, the SL RNP. Although the U snRNA requirements for trans-splicing are fairly well understood, little is known about the nature or function of protein factors that must participate in this processing reaction. Preliminary studies have revealed that protein constituents of the SL RNP particle include core Sm proteins and four specific proteins. These specific proteins are, in addition to the SL RNA itself, the only known components of the splicing apparatus that are unique to trans-splicing. The proposed research addresses five specific questions: 1) What is the primary sequence of each SL RNP specific protein, and are homologous proteins present in other organisms that carry out SL addition trans-splicing? 2) Are individual specific proteins required for function and if so, at what stage of splicing do they function? 3) Do the SL-specific proteins interact with other trans-spliceosomal components? 4) How is the SL RNP assembled and how do the specific proteins interact with the Sm core proteins? and 5) Does the Stem II region of the SL RNA bind protein or RNA factors in the trans-spliceosome and why doesn't U1 snRNP recognize the SL RNA's 5, splice site? In combination, the proposed studies should provide fundamental new insight into the mechanism of trans- splicing, how this process differs from cis-splicing and how complex ribonucleoprotein particles assemble and function. Finally, although trans- splicing is neither an adaptation to parasitism nor restricted to parasitic organisms, this RNA processing pathway is present in a wide variety of parasites with medical, veterinary and agricultural importance. A thorough understanding of trans-splicing, particularly how it differs biochemically from cis-splicing, may suggest possible avenues for therapeutic intervention in diseases caused by these pathogens.
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