Cap-interacting proteins in metazoan trans-splicing

描述（由申请人提供）：剪接前导（SL）RNA反式剪接通过将剪接前导序列添加到前体mRNA的5'末端来产生mRNA的成熟5'末端。反式剪接是线虫基因表达的重要机制。后生动物反式剪接的一个独特方面是，剪接的前导序列的添加也带来了一个新的和非典型的帽的mRNA，三甲基鸟苷帽（m2，2，7 GpppN）相比，典型的m7 GpppN真核生物帽。两个mRNA群体共存于线虫中：1）具有典型m7 GpppN帽和可变5'末端序列的非反式剪接，和2）具有m2，2，7 GpppN帽和共同5' 22 nt剪接前导序列的反式剪接。哺乳动物mRNA仅获得m7 G-cap。细胞帽相互作用蛋白介导反式剪接mRNA的代谢，并且对于翻译以及因此线虫基因表达是必需的。翻译起始因子eIF 4 E直接结合mRNA帽。这是大多数mRNA向核糖体募集的关键和限速步骤，并且是翻译控制的主要靶标。线虫eIF 4 E如何适应这两个RNA群体的翻译仍然是一个重要的，未回答的问题。 线虫感染地球上几乎一半的人（约30亿人），蛔虫感染约10亿人。由于线虫中的mRNA翻译必须不同于哺乳动物宿主，反式剪接的mRNA的翻译为药物开发提供了有吸引力的靶标。我们已经确定了蛔虫mRNA翻译机制的关键特征：1）蛔虫eIF 4 E同种型启动反式剪接和非反式剪接线虫mRNA的翻译，但与m7 G-帽相比，对m2，2，7 G-的亲和力低得多; 2）具有m2，2，7 G-帽的mRNA的翻译需要SL内的茎环和特异性序列，其对于m2，2，7 G加帽mRNA（“SL效应”）; 3）m2，2，7 G-SL mRNA的有效翻译需要在蛔虫eIF 4 E-3和eIF 4G翻译起始蛋白中的适应; 4）我们确定了结合到两个不同帽的蛔虫eIF 4 E-3的晶体结构，并定义了eIF 4 E在结合两个帽和SL时的NMR构象变化;和5）蛔虫有几种eIF 4 E同种型，它们翻译两种类型的mRNA，我们假设它们翻译不同的mRNA子集。我们的研究现在使我们能够机械地表征“SL效应”和线虫翻译。我们的目标是了解SL序列如何促进m2，2，7 G-加帽的mRNA的翻译。我们将通过1）确定蛔虫eIF 4 E如何与m2，2，7 G-SL相互作用，2）确定SL促进m2，2，7 G-加帽mRNA翻译的机制，3）确定线虫m2，2，7 G-SL和m2，2，7 G-SL-eIF 4 E复合物的结构，和4）确定蛔虫eIF 4 E同种型在不同蛔虫mRNA翻译中的作用。这些分析有望为线虫基因表达机制和翻译机制对被认为是“重大被忽视的疾病”的一组重要寄生虫中的反式剪接的适应提供重要的见解。在这些研究的结论中，我们期望更好地理解蛔虫eIF 4 E如何翻译m2，2，7 G-加帽的mRNA，蛔虫eIF 4 E如何与反式剪接的SL茎环相互作用，m2，2，7 G-SL和m2，2，7 G-SL-eIF 4 E复合物的结构，以及在重要的人类寄生虫中在mRNA翻译中起作用的蛋白质的潜在作用。此外，我们的研究将提供一般的洞察翻译起始，eIF 4 E异构体，和5' UTR元件在mRNA翻译中的作用，这将有广泛的影响，在其他真核生物的翻译。

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.