Diversity in cap-binding translation factor complexes: roles in translation initiation and mechanisms of mRNA selection

帽结合翻译因子复合物的多样性：翻译起始中的作用和 mRNA 选择机制

• 批准号: 419208155
• 负责人: Professorin Dr. Christine Elizabeth Clayton
• 金额: --
• 依托单位: Zentrum für Molekulare Biologie (ZMBH)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/419208155?language=en
• 关键词: Diversity; cap; binding; translation; factor

Eukaryotic translation initiation starts when eIF4E binds to the cap at the 5'-end of the mRNA. eIF4E recruits eIF4G, which binds more translation factors and the ribosome. Many eukaryotic organisms have several different EIF4Es that are active in translation initiation, but in no case are their individual roles known. We wish to answer this question using Trypanosoma brucei as a model system.Two trypanosome life-cycle stages grow in vitro: bloodstream forms (from mammals) and procyclic forms (from Tsetse flies). Trypanosomes are a good experimental model because nearly all gene regulation is post-transcriptional, mRNAs show large variations in mRNAs translation efficiencies and half-lives, and experimental manipulation is fast and convenient. T. brucei has 6 eIF4Es and 5 eIF4Gs. The EIF4E3-EIF4G4 and EIF4E4-EIF4G3 complexes are responsible for most initiation. EIF4E1 has no EIF4G partner; it is an inhibitor when bound to 4EIP. EIF4E2 interacts with SLBP2, an RNA-binding protein (RBP), but not with any EIF4G. This application concerns mainly three complexes that seem likely to have specialized roles in translation initiation: EIF4E5-EIF4G1, EIF4E5-EIF4G2, and EIF4E6-EIF4G5.De Melo Neto focuses on translation initiation factors of T. brucei and Leishmania, and has many relevant plasmids, pure recombinant proteins and antibodies. Clayton specializes in regulation of mRNA decay and translation by RBPs in T. brucei. In preparation we will identify all proteins that are associated with each EIF4E in bloodstream forms, either directly or via mRNA: the proteomes of the messenger ribonucleoprotein complexes (mRNPs).We hypothesise that individual EIF4E-G complexes are recruited to mRNAs by sequence-specific RBPs. Procyclic-form EIF4E2, EIF4E5 and EIF4E6 complexes are known to have some directly-associated RBPs, and we expect to find more in the mRNP proteomes. Using purified proteins, interactions within EIF4E5 and EIF4E6 complexes will be characterized. We will identify the mRNAs bound both by EIF4E2, EIF4E5 and EIF4E6 complexes and by selected associated RBPs. Meanwhile we will measure translation initiation and elongation, and poly(A) tail lengths, for all mRNAs in bloodstream forms. From the results we will be able to see, for each EIF4E complex, the characteristics of the bound mRNAs; and also which of these mRNAs might be recruited by particular RBPs. We then expect to focus on EIF4E5 and EIF4E6. We will measure the abundance and polysome association of bound mRNAs in procyclic and bloodstream forms depleted of relevant complex components and RBPs, and assess transcriptome-wide effects on mRNA abundance and translation.The work will reveal the roles of specialized translation initiation complexes, how they are recruited to specific mRNAs, and the effects of their recruitment on translation. In addition we will gain more insight into links between translation initiation, elongation, and mRNA decay.

当eIF4E结合到mRNA的5'端时，真核生物的翻译起始就开始了。eIF4E招募eIF4G，后者结合更多的翻译因子和核糖体。许多真核生物有几种不同的eif4e，它们在翻译起始中活跃，但在任何情况下都不知道它们各自的作用。我们希望用布鲁氏锥虫作为模型系统来回答这个问题。两个锥虫生命周期阶段在体外生长：血流形式（来自哺乳动物）和前循环形式（来自采采蝇）。锥虫是一个很好的实验模型，因为几乎所有的基因调控都是转录后的，mrna的翻译效率和半衰期变化很大，实验操作快捷方便。布鲁氏有6个eif4e和5个eIF4Gs。EIF4E3-EIF4G4和EIF4E4-EIF4G3复合物负责大部分起始。EIF4E1没有EIF4G合作伙伴；当与4EIP结合时，它是一种抑制剂。EIF4E2与rna结合蛋白（RBP） SLBP2相互作用，但不与EIF4G相互作用。该应用程序主要涉及三种复合物，它们似乎可能在翻译起始中起特殊作用：EIF4E5-EIF4G1， EIF4E5-EIF4G2和EIF4E6-EIF4G5。De Melo Neto主要研究布鲁氏杆菌和利什曼原虫的翻译起始因子，有许多相关的质粒、纯重组蛋白和抗体。Clayton专门研究布鲁氏T. brucei中rbp对mRNA衰变和翻译的调控。在准备过程中，我们将识别与血流形式中每个EIF4E相关的所有蛋白质，直接或通过mRNA：信使核糖核蛋白复合物（mRNPs）的蛋白质组。我们假设单个EIF4E-G复合物是通过序列特异性rbp招募到mrna的。已知proccyclic -form EIF4E2， EIF4E5和EIF4E6复合物具有一些直接相关的rbp，我们希望在mRNP蛋白质组中发现更多。使用纯化蛋白，将表征EIF4E5和EIF4E6复合物内的相互作用。我们将鉴定与EIF4E2， EIF4E5和EIF4E6复合物以及选定的相关rbp结合的mrna。同时，我们将测量血液形式中所有mrna的翻译起始和延伸，以及聚(A)尾长度。从结果中我们将能够看到，对于每个EIF4E复合物，结合的mrna的特征；以及哪些mrna可能被特定的rbp招募。然后我们期望关注EIF4E5和EIF4E6。我们将在缺乏相关复杂成分和rbp的前环和血流形态中测量结合mRNA的丰度和多体关联，并评估转录组范围内对mRNA丰度和翻译的影响。这项工作将揭示专门的翻译起始复合物的作用，它们如何被招募到特定的mrna上，以及它们的招募对翻译的影响。此外，我们将更深入地了解翻译起始、延伸和mRNA衰变之间的联系。