Cap-interacting proteins in metazoan trans-splicing

后生动物转拼中的帽相互作用蛋白

• 批准号: 8395859
• 负责人: RICHARD E. DAVIS
• 金额: $ 39.33万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8395859
• 关键词: 5' Untranslated Regions; Affinity; Area; Ascaris; Award; Binding; Cell-Free System; Cells; Complex; Data; Development; Disease; Drug Delivery Systems; Embryo; Eukaryota; Exhibits; Funding; Gene Expression; Genetic Translation; Goals; Helminths; Human; Immunoprecipitation; In Vitro; Indium; Intestines; Knowledge; Lead; Mediating; Messenger RNA; Metabolism; Methods; NMR Spectroscopy; Nematoda; Organism; Parasites; Parasitic nematode; Peptide Initiation Factors; Play; Population; Process; Protein Isoforms; Proteins; Public Health; RNA; RNA Caps; RNA Sequences; RNA Splicing; Ribosomes; Role; Spliced Leader RNA; Spliced Leader Sequences; Structure; System; Testis; Trans-Splicing; Transfection; Translating; Translation Initiation; Translations; Untranslated Regions; X-Ray Crystallography; base; drug development; in vivo; insight; mRNA Decay; mRNA Precursor; mRNA capping; neglect; novel; novel therapeutics; particle; protein function; reconstitution; socioeconomics; stem; tool; translation factor

DESCRIPTION (provided by applicant): Spliced leader (SL) RNA trans-splicing generates the mature 5' ends of mRNAs by addition of a spliced leader sequence to the 5' end of a pre-mRNA. Trans-splicing is an essential mechanism of gene expression in nematodes. A unique aspect of metazoan trans-splicing is that addition of the spliced leader sequence also brings a new and atypical cap to the mRNA, a trimethylguanosine cap (m2,2,7GpppN) compared to the typical m7GpppN eukaryotic cap. Two populations of mRNAs co-exist in nematodes: 1) non-trans-spliced with a typical m7GpppN cap and variable 5' end sequence and 2) trans- spliced with an m2,2,7GpppN cap and a common 5' 22 nt spliced leader sequence. Mammalian mRNAs only acquire an m7G-cap. Cellular cap-interacting proteins mediate the metabolism of trans-spliced mRNAs and are essential for translation and, therefore, nematode gene expression. The translation initiation factor eIF4E directly binds the mRNA cap. This is the critical and rate limiting step in recruitment of most mRNAs to the ribosome and is a major target for translational control. How nematode eIF4E has adapted to accommodate translation of these two RNA populations remains an important, unanswered question. Nematodes infect almost half the people on earth (~3 billion people) and Ascaris infects ~1 billion people. As mRNA translation in nematodes must differ from the mammalian host, translation of trans- spliced mRNAs provides an attractive target for drug development. We have identified key features of the mechanism of translation of Ascaris mRNAs: 1) Ascaris eIF4E isoforms initiate translation of both trans- spliced and non-trans-spliced nematode mRNAs, yet exhibit a much lower affinity for the m2,2,7G- compared to the m7G-cap; 2) Translation of mRNAs with a m2,2,7G-cap requires a stem loop and specific sequences within the SL that are necessary and sufficient for efficient translation of m2,2,7G-capped mRNAs (the "SL effect"); 3) Efficient translation of the m2,2,7G-SL mRNAs requires adaptations in Ascaris eIF4E-3 and eIF4G translation initiation proteins; 4) We determined the crystal structures of Ascaris eIF4E-3 bound to the two different caps and defined NMR conformational changes in eIF4E on binding the two caps and SL; and 5) Ascaris has several eIF4E isoforms that translate both types of mRNAs and we hypothesize they translate distinct subsets of mRNAs. Our studies now enable us to mechanistically characterize the "SL Effect" and nematode translation. We aim to understand how the SL sequence facilitates translation of m2,2,7G-capped mRNAs. We will pursue this goal by 1) determining how Ascaris eIF4E interacts with the m2,2,7G-SL, 2) determining the mechanism(s) through which the SL facilitates translation of m2,2,7G-capped mRNAs, 3) determining the structure of the nematode m2,2,7G-SL and m2,2,7G-SL-eIF4E complex, and 4) determine the role of Ascaris eIF4E isoforms in the translation of different Ascaris mRNAs. These analyses promise to provide important insights into mechanisms of nematode gene expression and adaptation of the translation machinery to trans-splicing in an important group of parasites considered to be "Great Neglected Diseases". At the conclusion of these studies, we expect to have a better understanding of how Ascaris eIF4E translates m2,2,7G -capped mRNAs, how Ascaris eIF4E interacts with the trans-spliced SL stem-loop, the structure of the m2,2,7G -SL and m2,2,7G - SL-eIF4E complex, and the potential role of proteins that function in mRNA translation in an important human parasite. Moreover, our studies will provide general insight into translation initiation, eIF4E isoforms, and the role of the 5' UTR element in mRNA translation that will have broad implications for translation in other eukaryotes. PUBLIC HEALTH RELEVANCE: Parasitic nematodes remain a significant public health problem in many parts of the world. Ascaris alone infects upwards of 1 billion people and hinders socioeconomic development in endemic areas. We will carry out studies on novel cap-interacting proteins in Ascaris that are potential targets for new and novel therapeutics against parasitic helminths.

描述（由申请人提供）：剪接的引导者（SL）RNA transplicing通过在前MRNA的5'端添加剪接的引导序列，从而生成mRNA的成熟5'端。跨跨性是线虫中基因表达的基本机制。后生式移植的一个独特方面是，与典型的M7GPPPN真核生物帽相比，添加剪接的引导序列还为mRNA带来了一个新的和非典型的帽，这是一种三甲基鸟苷帽（M2,2,7GPPPN）。线虫中的两个mRNA群体共存：1）与典型的M7GPPPN CAP和可变的5'末端序列和2）用M2,2,7GPPPN CAP和常见的5'22 NT固定领导者序列进行反式剪接的非变型。哺乳动物mRNA只能获得M7G-CAP。细胞帽相互作用的蛋白介导移植的mRNA的代谢，因此对于翻译至关重要，因此是线虫基因表达。翻译起始因子EIF4E直接结合mRNA帽。这是将大多数mRNA募集到核糖体中的关键和速率限制步骤，并且是转化控制的主要目标。线虫EIF4E如何适应这两个RNA种群的翻译仍然是一个重要的，未解决的问题。 线虫感染了地球上几乎一半的人（约30亿人），阿斯卡里斯感染了约10亿人。由于线虫中的mRNA翻译必须与哺乳动物宿主不同，因此剪接mRNA的翻译为药物发育提供了有吸引力的靶标。我们已经确定了Ascaris mRNA翻译机理的关键特征：1）Ascaris eIF4E同工型启动了跨剪接和非反trans型线虫mRNA的翻译，但与M7G-CAP相比，M2,2,7G-的亲和力低得多。 2）用M2,2,7G-CAP的mRNA翻译需要SL内的茎环和特定序列，足以有效地翻译M2,2,7G盖的mRNA（“ SL效应”）； 3）M2,2,7g-SL mRNA的有效翻译需要在Ascaris EIF4E-3和EIF4G转换蛋白质中进行适应； 4）我们确定了与两个不同盖结合的ascaris eif4e-3的晶体结构，并定义了EIF4E中的NMR构象变化，即结合两个盖子和SL； 5）asscaris具有几种EIF4E同工型，可以翻译两种类型的mRNA，我们假设它们翻译了mRNA的不同子集。现在，我们的研究使我们能够机械地表征“ SL效应”和线虫翻译。我们旨在了解SL序列如何促进M2,2,7G封闭的mRNA的翻译。我们将通过1）确定Ascaris EIF4E如何与M2,2,7G-SL相互作用，2）确定SL促进M2,2,7G盖的mRNA的转换的机制，3）确定线虫的结构M2 M2,2,7G-SL和M2,2,2,7G-SL-eff4ECTRAME的结构EIF4E在不同assaris mRNA的翻译中的同工型。这些分析有望提供对线虫基因表达机制的重要见解，并在一组被认为是“大被忽视的疾病”的重要寄生虫中翻译机制对跨拼层的适应。在这些研究的结论中，我们希望更好地了解Ascaris EIF4E如何翻译M2,2,7g封闭的mRNA，Ascaris EIF4E如何与移植的SL茎环相互作用，M2,2,7G -SL和M2,2,7G -SL -EIF4E复合物的结构（M2,2,7g -SL和M.2,7g -SL和SL -EIFCONCELS的结构）的作用，以及一个重要的角色，以及一定的角色，该作用在某种程度上构成了一定功能。此外，我们的研究将提供对翻译起始，EIF4E同工型的一般见解，以及5'UTR元素在mRNA翻译中的作用，这将对其他真核生物的翻译具有广泛的影响。 公共卫生相关性：寄生线虫在世界许多地方仍然是一个重大的公共卫生问题。仅阿斯卡里斯（Ascaris）就会感染超过10亿人，并阻碍了地方性地区的社会经济发展。我们将对阿斯卡里斯的新型帽相互作用蛋白进行研究，这些蛋白质是针对寄生虫蠕虫的新型和新型疗法的潜在靶标。