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

当eIF4E与mRNA 5‘端的帽子结合时，真核细胞的翻译启动就开始了。EIF4E招募了eIF4G，它绑定了更多的翻译因子和核糖体。许多真核生物具有几种不同的EIF4E，它们活跃在翻译起始过程中，但它们的单独作用尚不清楚。我们希望以布氏锥虫为模型系统来回答这个问题。体外生长有两个锥虫生命周期阶段：血流形式(来自哺乳动物)和原环状形式(来自采采蝇)。锥体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，mRNAs在mRNAs翻译效率和半衰期方面表现出很大的差异，并且实验操作快速方便。布氏锥虫有6个eIF4E和5个eIF4G。EIF4E3-EIF4G4和EIF4E4-EIF4G3复合体是引发反应的主体。EIF4E1没有EIF4G合作伙伴；当绑定到4EIP时，它是一个抑制物。EIF4E2与RNA结合蛋白SLBP2相互作用，但不与任何EIF4G相互作用。这一应用主要涉及三个可能在翻译起始中具有特殊作用的复合体：EIF4E5-EIF4G1、EIF4E5-EIF4G2和EIF4E6-EIF4G5。DMelo Neto专注于布氏毛滴虫和利什曼原虫的翻译起始因子，并具有许多相关的质粒、纯重组蛋白和抗体。克莱顿专门研究布氏毛滴虫中限制性商业惯例对信使核糖核酸的降解和翻译。在准备过程中，我们将鉴定与每个eIF4E以血流形式存在的所有蛋白质，无论是直接还是通过mRNA：信使核糖核蛋白复合体(MRNPs)的蛋白质组。我们假设个别的eIF4E-G复合体通过序列特异性限制性商业惯例被招募到mRNAs。已知前环型EIF4E2、EIF4E5和EIF4E6复合体具有一些直接相关的限制性商业惯例，我们希望在mRNP蛋白质组中发现更多。使用纯化的蛋白质，将表征EIF4E5和EIF4E6复合体之间的相互作用。我们将确定EIF4E2、EIF4E5和EIF4E6复合体以及选定的相关限制性商业惯例结合的mRNAs。同时，我们将测量所有血流形式的mRNAs的翻译起始和延伸，以及Poly(A)尾长。从结果中，我们将能够看到，对于每个eIF4E复合体，结合的mRNAs的特征；以及这些mRNAs中的哪些可能被特定的限制性商业惯例招募。然后，我们将重点介绍EIF4E5和EIF4E6。我们将测量缺乏相关复杂成分和限制性商业惯例的原循环和血流形式中结合的mRNAs的丰度和多聚体结合，并评估整个转录组对mRNA量和翻译的影响。工作将揭示专门的翻译起始复合体的作用，它们如何被招募到特定的mRNAs，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、延伸和信使核糖核酸衰变之间的联系。

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.