Cap-interacting proteins in metazoan trans-splicing

描述（由申请人提供）：据估计，肠道线虫感染了约30亿人。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。线虫基因表达知之甚少。反式剪接是寄生线虫中基因表达的关键机制，因为其超过70%的mRNA通过反式剪接进行成熟。在线虫中，mRNA代谢和帽相互作用蛋白必须处理两个群体的mRNA，剪接的前导反式剪接的mRNA与三甲基鸟苷帽和非反式剪接的mRNA与单甲基鸟苷帽。我们鉴定并初步研究了线虫中的几种帽相互作用蛋白，包括翻译起始因子eIF4E和两种脱帽蛋白DcpS和Dcp2。eIF4E和DcpS在它们的底物特异性方面不同于它们的哺乳动物对应物。具体而言，它们可以与单甲基和三甲基鸟苷帽相互作用，而哺乳动物形式仅与单甲基帽相互作用。帽下游的反式剪接的剪接前导序列增强eIF4E与三甲基鸟苷帽的帽结合和翻译，而它减少mRNA的Dcp2脱帽。这些蛋白质表现出线虫基因表达所需的独特性质。这项建议的总体目标是确定这些帽相互作用蛋白如何有助于线虫的mRNA代谢。我们将使用蛔虫胚胎无细胞系统来更好地理解几种eIF4E同种型在mRNA翻译中的作用。还将进行研究以调查调节这些蛋白质活性的拼接前导序列的序列或结构属性以及实现这些独特相互作用的蛋白质决定簇。我们将这些帽相互作用的蛋白质的特点，就其底物特异性和调查的蛋白质的决定因素，使这些蛋白质与三甲基鸟苷帽不像他们的哺乳动物宿主相互作用。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，可能导致进一步的研究，允许开发针对广谱线虫寄生虫的新的驱虫剂。

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.