Cap-interacting proteins in metazoan trans-splicing

描述（由申请人提供）：据估计，肠道线虫感染的人口高达30亿。这些寄生虫的社会经济影响是相当大的，它们阻碍了许多地区的医疗和经济改善。人们对线虫的基因表达了解甚少。反式剪接是寄生线虫基因表达的一个关键机制，超过70%的寄生线虫mrna通过反式剪接成熟。在线虫中，mRNA代谢和帽层相互作用蛋白必须处理两种mRNA群体，具有三甲基鸟苷帽的剪接先导反式剪接mRNA和具有单甲基鸟苷帽的非反式剪接mRNA。我们鉴定并开展了线虫中几种帽层相互作用蛋白的初步研究，包括翻译起始因子elF4E和两个去帽蛋白，dps和Dcp2。elF4E和DcpS在底物特异性上不同于哺乳动物。具体来说，它们可以与单甲基和三甲基鸟苷帽相互作用，而哺乳动物形式只与单甲基帽相互作用。帽下游的反式剪接前导序列增强了elF4E与三甲基鸟苷帽的帽结合和翻译，而减少了mRNA的Dcp2去帽。这些蛋白质表现出线虫基因表达所需要的独特特性。本提案的总体目标是确定这些帽相互作用蛋白如何促进线虫的mRNA代谢。我们将使用蛔虫胚胎无细胞系统来更好地了解几种elF4E亚型在mrna翻译中的作用。研究还将开展调查的序列或结构属性的剪接先导序列，调节这些蛋白质的活性和蛋白质决定因素，使这些独特的相互作用。我们将对这些帽相互作用蛋白的底物特异性进行表征，并研究使这些蛋白与哺乳动物宿主不同的三甲基鸟苷帽相互作用的蛋白质决定因素。这些研究将深入了解寄生线虫基因表达的基本方面以及帽相互作用蛋白在mRNA代谢中的作用。这些研究有可能确定新的药物靶点，从而导致进一步的研究，从而开发针对广泛的线虫寄生虫的新驱虫药。

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.