Abstract 2083 Exploring the role of 5′-UTRs in mRNA sensitivity to eIF3 mutations

Translation by the ribosome is required for cell growth, development, and response to external stimuli.Translation is primarily regulated at the initiation phase, which begins when several eukaryotic initiation factors (eIFs) load onto the small ribosomal (40S) subunit to form a pre-initiation complex (PIC).The PIC is then thought to bind the 5 0 end of mRNA in an open conformation that enables mRNA loading and subsequent scanning through the 5 0 -untranslated region (5 0 -UTR) until it locates the start codon.Upon start-codon recognition, the PIC closes to arrest scanning.mRNA attachment, scanning, and start-codon recognition together comprise mRNA recruitment, the defining feature of translation initiation in eukaryotes.eIF3 is a multisubunit complex that is required for mRNA recruitment.And yet, how eIF3 or its constituent subunits contribute mechanistically to mRNA recruitment remains unknown.We leverage two eIF3 mutations that target either the entire complex (eIF3a/b Degron) or disrupt binding of two of its subunits (eIF3i/g) to the remainder of the complex (eIF3i DDKK).This latter mutation severely disrupts regions of eIF3 that bind near the mRNA-entry-channel (mEnC) of the ribosome.Using ribosome profiling, we have identified specific mRNAs whose translation is particularly sensitive to disruption of either the full eIF3 complex or its mEnC arm.However, it remains unclear which specific mRNA sequences drive this sensitivity.To ask whether the 5 0 UTRs of these sensitive mRNAs are responsible for these effects, we have constructed Luciferase reporter plasmids containing the 5 0 UTR and first 20 codons of sensitive mRNAs and have begun testing whether these regions disrupt Luciferase expression in eIF3a/b Degron and eIF3i DDKK cells.These experiments will illuminate the role of 5 0 -UTR sequences in conferring sensitivity to disruption of eIF3 or its mEnC arm, or whether other sequences within an mRNA also contribute to these effects.