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: 8862341
• 负责人: RICHARD E. DAVIS
• 金额: $ 41.67万
• 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 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8862341
• 关键词: 5' Untranslated Regions; Affinity; Area; Ascaris; Award; Binding; Cell-Free System; Cells; Complex; Data; Development; Disease; Drug Targeting; 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; transcriptome sequencing; 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.

描述（由申请人提供）：剪接前导序列（SL）RNA反式剪接通过将剪接前导序列添加到前mRNA的5'端来产生mRNA的成熟5'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，与典型的 m7GpppN 真核帽相比，添加剪接前导序列还为 mRNA 带来新的非典型帽，即三甲基鸟苷帽 (m2,2,7GpppN)。线虫中共存有两个 mRNA 群体：1) 具有典型 m7GpppN 帽和可变 5' 末端序列的非反式剪接，以及 2) 具有 m2,2,7GpppN 帽和常见 5' 22 nt 剪接的反式剪接前导序列。哺乳动物 mRNA 仅获得 m7G 帽。细胞帽相互作用蛋白介导转剪 mRNA 的代谢，对于翻译以及线虫基因表达至关重要。翻译起始因子 eIF4E 直接结合 mRNA 帽。这是大多数 mRNA 募集到核糖体中的关键和限速步骤，也是翻译控制的主要目标。线虫 eIF4E 如何适应这两个 RNA 群体的翻译仍然是一个重要的、尚未解答的问题。 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 翻译必须与哺乳动物宿主不同，因此转拼 mRNA 的翻译为药物开发提供了一个有吸引力的目标。我们已经确定了蛔虫 mRNA 翻译机制的关键特征：1) 蛔虫 eIF4E 同种型启动转拼和非反剪接线虫 mRNA 的翻译，但与 m2,2,7G- 相比，表现出低得多的亲和力。到 m7G 帽； 2) 具有 m2,2,7G-cap 的 mRNA 的翻译需要 SL 内的茎环和特定序列，这些序列对于有效翻译 m2,2,7G-cap 的 mRNA 是必要且充分的（“SL 效应”）； 3) m2,2,7G-SL mRNA 的有效翻译需要对蛔虫 eIF4E-3 和 eIF4G 翻译起始蛋白进行调整； 4) 我们确定了与两个不同帽结合的蛔虫 eIF4E-3 的晶体结构，并定义了 eIF4E 在结合两个帽和 SL 时的 NMR 构象变化； 5) 蛔虫有几种翻译两种类型 mRNA 的 eIF4E 同工型，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从机制上表征“SL 效应”和线虫翻译。我们的目标是了解 SL 序列如何促进 m2,2,7G 加帽 mRNA 的翻译。我们将通过以下方式实现这一目标：1) 确定蛔虫 eIF4E 如何与 m2,2,7G-SL 相互作用，2) 确定 SL 促进 m2,2,7G-capped mRNA 翻译的机制，3) 确定线虫 m2,2,7G-SL 和 m2,2,7G-SL-eIF4E 复合物的结构，4) 确定蛔虫 eIF4E 的作用不同蛔虫 mRNA 翻译中的亚型。这些分析有望为线虫基因表达机制以及翻译机制对被认为是“被忽视的重大疾病”的重要寄生虫群中反式剪接的适应机制提供重要见解。在这些研究的结论中，我们希望更好地了解蛔虫 eIF4E 如何翻译 m2,2,7G 加帽的 mRNA，蛔虫 eIF4E 如何与反式剪接的 SL 茎环相互作用，m2,2, 7G -SL 和 m2,2,7G - SL-eIF4E 复合物，以及在重要人类寄生虫的 mRNA 翻译中起作用的蛋白质的潜在作用。此外，我们的研究将提供对翻译起始、eIF4E 同工型以及 5'UTR 元件在 mRNA 翻译中的作用的一般见解，这将对其他真核生物中的翻译产生广泛的影响。

项目成果

Structural requirements for Caenorhabditis elegans DcpS substrates based on fluorescence and HPLC enzyme kinetic studies.

• DOI: 10.1111/j.1742-4658.2010.07709.x
• 发表时间: 2010-07
• 期刊: The FEBS journal
• 作者: Wypijewska A;Bojarska E;Stepinski J;Jankowska-Anyszka M;Jemielity J;Davis RE;Darzynkiewicz E
• 通讯作者: Darzynkiewicz E

Small RNA pathways in the nematode Ascaris in the absence of piRNAs.

• DOI: 10.1038/s41467-022-28482-7
• 发表时间: 2022-02-11
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Zagoskin MV;Wang J;Neff AT;Veronezi GMB;Davis RE
• 通讯作者: Davis RE

5'-Terminal chemical capping of spliced leader RNAs.

剪接前导 RNA 的 5-末端化学加帽。

• DOI: 10.1016/j.tetlet.2012.06.127
• 发表时间: 2012
• 期刊: Tetrahedron letters
• 影响因子: 1.8
• 作者: Piecyk,Karolina;Davis,RichardE;Jankowska-Anyszka,Marzena
• 通讯作者: Jankowska-Anyszka,Marzena

Synthesis of N²-modified 7-methylguanosine 5'-monophosphates as nematode translation inhibitors.

• DOI: 10.1016/j.bmc.2012.05.078
• 发表时间: 2012-08-01
• 期刊: BIOORGANIC & MEDICINAL CHEMISTRY
• 影响因子: 3.5
• 作者: Piecyk, Karolina;Davis, Richard E.;Jankowska-Anyszka, Marzena
• 通讯作者: Jankowska-Anyszka, Marzena

Silencing of germline-expressed genes by DNA elimination in somatic cells.

• DOI: 10.1016/j.devcel.2012.09.020
• 发表时间: 2012-11-13
• 期刊: Developmental cell
• 影响因子: 11.8
• 作者: Wang J;Mitreva M;Berriman M;Thorne A;Magrini V;Koutsovoulos G;Kumar S;Blaxter ML;Davis RE
• 通讯作者: Davis RE