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

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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.