Abstract 1780 Disentangling the role of eIF3 and its subunits on the ribosome and across the transcriptome

Initiation is the most regulated phase of translation in eukaryotes. This multi-step pathway is mediated by numerous protein initiation factors (eIFs), of which eIF3 is the largest and most complex. eIF3 contributes to events throughout the initiation pathway, in particular playing critical roles in mRNA recruitment, a process which encompasses initial ribosome docking on an mRNA, scanning, and start-codon recognition. Recently, eIF3 has been implicated in driving the selective translation of specific classes of mRNAs in higher eukaryotes. Unraveling the mechanism of these diverse contributions - and disentangling the roles of the individual subunits of the eIF3 complex - nonetheless remains challenging, in part because all five subunits of the core complex are essential. We have employed ribosome profiling of budding yeast cells expressing two distinct mutations targeting the eIF3 complex. These mutations either disrupt the entire complex or subunits positioned near the mRNA-entry channel of the ribosome. Recent cryo-EM structures suggest that these mRNA-entry channel arm subunits relocate during or in response to mRNA binding and start-codon recognition. Our experiments show that disruption of either the entire eIF3 complex or specific targeting of its mRNA-entry-channel arm affect the translation of mRNAs with long 5'-UTRs and whose translation is more dependent on eIF4A, eIF4B, and Ded1 but less dependent on eIF4G, eIF4E, and PABP. Disruption of the entire eIF3 complex further affects mRNAs involved in mitochondrial processes and with more structured 5'-UTRs. To complement these efforts, we have employed tandem mass tag mass spectrometry (TMT-MS) and long-read nano pore sequencing of total and fractionated RNAs to monitor the effects of these mutations on the proteome and on specific mRNA isoforms across the transcriptome. In collaboration with the laboratory of Ruben Gonzalez, we also have developed a recombinantly-reconstituted eIF3 complex that recapitulates the functions of natively purified eIF3 in vitro, enabling its full mechanistic dissection in vitro using a suite of ensemble and single-molecule techniques. This work is supported by NIH/NIGMS R15 GM140372-01.