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

当eIF 4 E与mRNA 5 '端的帽结合时，真核翻译起始。eIF 4 E募集eIF 4G，eIF 4G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EIF 4 E，它们在翻译起始中具有活性，但在任何情况下都不知道它们各自的作用。我们希望用布氏锥虫作为模型系统来回答这个问题。锥虫在体外生长有两个生命周期阶段：血流型（来自哺乳动物）和原环型（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在mRNA翻译效率和半衰期方面表现出很大的变化，并且实验操作快速方便。T.布氏杆菌有6个eIF 4 E和5个eIF 4G。EIF 4 E3-EIF 4G 4和EIF 4 E4-EIF 4G 3复合物负责大部分起始。EIF 4 E1没有EIF 4G伴侣;当与4 EIP结合时，它是一种抑制剂。EIF 4 E2与RNA结合蛋白（RBP）SLBP 2相互作用，但不与任何EIF 4G相互作用。本申请主要涉及三种可能在翻译起始中起特殊作用的复合物：EIF 4 E5-EIF 4G 1、EIF 4 E5-EIF 4G 2和EIF 4 E6-EIF 4G 5。布氏杆菌和利什曼原虫的重组蛋白，并有许多相关的质粒、纯的重组蛋白和抗体。克莱顿专门研究T.布鲁塞。在准备过程中，我们将鉴定所有与血流形式的EIF 4 E相关的蛋白质，无论是直接的还是通过mRNA：信使核糖核蛋白复合物（mRNP）的蛋白质组。已知前环形式的EIF 4 E2、EIF 4 E5和EIF 4 E6复合物具有一些直接相关的RBP，我们期望在mRNP蛋白质组中发现更多。使用纯化的蛋白质，EIF 4 E5和EIF 4 E6复合物内的相互作用将被表征。我们将鉴定由EIF 4 E2、EIF 4 E5和EIF 4 E6复合物以及由选定的相关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poly（A）尾长度。从结果中，我们将能够看到，对于每个EIF 4 E复合物，结合的mRNA的特征;以及这些mRNA中的哪些可能被特定的RBP募集。然后，我们将重点关注EIF 4 E5和EIF 4 E6。我们将测量的丰度和多核糖体协会的结合mRNA的前循环和血流形式耗尽相关的复杂组件和RBP，并评估转录组范围内的mRNA丰度和translation.The工作将揭示专门的翻译起始复合物的作用，他们是如何招募到特定的mRNA，和他们的招聘翻译的影响。此外，我们将获得更多的洞察翻译起始，延长和mRNA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.