Cap-interacting proteins in metazoan trans-splicing

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DESCRIPTION (provided by applicant): It is estimated that intestinal nematodes infect upwards of ~3 billion people. The socioeconomic effects of these parasites are considerable and they hinder medical and economic improvement in many areas. Nematode gene expression is poorly understood. Trans-splicing is a key mechanism of gene expression in parasitic nematodes as > than 70% of their mRNAs undergo maturation by trans-splicing. In nematodes, mRNA metabolism and cap-interacting proteins must deal with two populations of mRNAs, spliced leader trans-spliced mRNAs with a trimethylguanosine cap and non-trans-spliced mRNAs with a monomethylguanosine cap. We identified and carried out initial studies on several cap-interacting proteins in nematodes including the translation initiation factor elF4E and two de-capping proteins, DcpS and Dcp2. elF4E and DcpS differ from their mammalian counterparts in their substrate specificity. Specifically, they can interact with both monomethyl and trimethylguanosine caps, whereas the mammalian forms only interact with monomethyl caps. The trans-spliced spliced leader sequence downstream of the cap enhances the cap- binding of elF4E to trimethylguanosine caps and translation, whereas it reduces Dcp2 de-capping of mRNA. These proteins exhibit unique properties that are required for nematode gene expression. The overall goal of this proposal is to determine how these cap-interacting proteins contribute to mRNA metabolism in nematodes. We will use an Ascaris embryo cell-free system to better understand the role of several elF4E isoforms in translation of mRNAs. Studies will also be carried out to investigate the sequence or structural attributes of the spliced leader sequence that modulate the activity of these proteins and the protein determinants that enable these unique interactions. We will characterize these cap-interacting proteins with respect to their substrate specificity and investigate the protein determinants that enable these proteins to interact with trimethylguanosine caps unlike their mammalian hosts. The studies will provide insight into essential aspects of gene expression in parasitic nematodes and the role of cap-interacting proteins in mRNA metabolism. These studies have the potential to identify novel drug targets that could lead to further studies allowing development of new anthelmintics against a broad spectrum of nematode parasites.