Abstract 1778 Leveraging a recombinantly-reconstituted eIF3 complex to dissect the contributions of its subunits to RNA binding

Eukaryotic translation initiation factor 3 (eIF3) is a multi-subunit complex that participates in events throughout translation initiation and has recently emerged as a player in translational regulation. eIF3 contributes to formation of the pre-initiation complex, in which the initiator tRNA and several initiation factors (eIFs) assemble on the small ribosomal subunit, and is required for the recruitment of mRNA to PIC. eIF3 also contributes to scanning of the 5'-UTR by the PIC and subsequent start-codon recognition. And yet, the mechanistic contributions of eIF3 and its subunits remain mysterious. The five subunits of the yeast complex are essential and previous work has relied on eIF3 purified natively from yeast. Thus, in vitro investigation has previously been limited to the study of viable functional variants. In collaboration with the laboratory of Ruben Gonzalez, we have developed a recombinantly-reconstituted eIF3 complex that recapitulates the functions of the natively purified complex in vitro. This system enables the systematic dissection of eIF3 and its subunits. Using a fluorometric assay, we are investigating RNA binding by the individual eIF3 subunits, as well as their binding to other subunits of the complex. We have focused our binding experiments on the mRNA-entry channel (mEnC) arm of eIF3, comprised of the eIF3a CTD, eIF3b, and eIF3i and eIF3g. We are working to determine which subunits of the mEnC arm contribute to RNA binding and to the overall integrity of the mEnC arm. This work is supported by NIH/NIGMS R15 GM140372-01.