eIF3-mediated ribosome recruitment by histone H4 mRNA during translation initiation – H4translation

Our lab has shown that histone H4 mRNA combines canonical features (cap-dependent translation) with viral strategy (lack of scanning and internal recruitment of initiation factors) during translation initiation (Mol. Cell, 2011). It contains a double stem-loop structure called eIF4E-sensitive element (4E-SE) that recruits the trimeric cap-binding complex eIF4F. In addition, H4 mRNA contains a three-way junction (TWJ) with the unusual property of stalling engaged 80S ribosomes when cap-dependent translation is repressed. Using cryo-EM, we also showed that the mRNA exhibits shortly after the start codon a nucleotide sequence complementary to helix 16 of 18S ribosomal RNA sequence that helps mRNA binding and proper AUG positioning. This was the first description of a cellular IRES-like structure on the ribosome (Nat. Commun., 2016; joint publication of partners 1 and 2). Recently, CLIP-SEQ experiments showed that H4 mRNA also binds the largest initiation factor eIF3. In higher eukaryotes, eIF3 subunits are grouped into core subunits (a, c, e, f, h, l, k, m) and peripheral subunits (b, d, g, i, j). Whereas the core module forms a distinctive multilobed structure conserved in the proteasome and signalosome complexes, the peripheral subunits of eIF3 are more dispersed, forming a network of interactions encircling the 40S subunit and connecting the entry of the mRNA channel to its exit. According to our preliminary experiments, histone H4 mRNA interacts with eIF3 at both ends of the mRNA channel where the eIF3b/g/i subcomplex and eIF3d subunit should be located. Building up on these findings, we propose the present H4translation research project. It consists of 3 tasks dedicated to assess the functional and structural features of the interaction of eIF3 with the 40S subunit during the initiation step on histone H4 mRNA and clarify the structural ambiguities concerning eIF3 subunit localization on the ribosome. (1) The first goal of the project is to decipher the eIF3-H4 mRNA interaction. Approaches here used will include RNA probing, foot-printing analysis, translation assays, and cross-linking. We will focus our attention on the eIF3d subunit which exhibits a cap-binding activity and could play a critical function for the H4 tethering mechanism. This task should provide a comprehensive evaluation of how eIF3 interacts with a folded mRNA structure such as H4. (2) The structural investigation is another important goal of the project. First, we will reconstitute a complex between H4 mRNA and the human ribosome and push the resolution to the 3-4 Å range in order to visualize the nucleotides involved in this unprecedented 18S ribosomal RNA interaction with an mRNA. In addition, we will adapt our purification protocol in order to prepare 48S complexes assembled in crude translation extracts and blocked on the H4 mRNA start codon. Several structures of incomplete 48S complexes have been reported in the literature, with only weak and partial occupancy of eIF3 peripheral subunits. We believe that histone H4 mRNA is an excellent platform for ribosome and eIF3 complex formation since it binds the factor with internal sequences and potentially with the 5’ cap. (3) In the third goal, we will explore the effect of the eIF3 interaction in vivo taking advantage of our expertise in mRNA silencing. Specific eIF3 subunits will be down-regulated in cell cultures by siRNA interference and the effect on histone H4 translation will be studied. The analysis will be extended to other mRNA targets previously identified by CLIP-SEQ in order to have a broader overview of the eIF3 regulation. Taken together, we expect fundamental structure-function insights into the IRES-like mechanism of cap-dependent internal translation initiation of H4 mRNA. In addition, the project will provide understanding on how eIF3 assembles within the full translation initiation complex and how its selects and regulates some characteristic cellular mRNAs.