Abstract 1807 Interrogating the role of eIF3 in translation initiation with transcript-isoform specificity

Translation is an essential biological process in which the sequence of a messenger RNA molecule is transformed into a functional protein. Initiation is the rate-limiting and most highly regulated stage of translation, sets the reading frame for protein synthesis, and thus represents one of the most important readings of the genetic code. Eukaryotic initiation factor 3 (eIF3) is a multi-subunit complex that participates in each phase of translation initiation. eIF3 contributes to formation of the pre-initiation complex containing initiator tRNA, eIFs, and the small ribosomal subunit. It also promotes mRNA attachment by the PIC, scanning, and start codon recognition. Altered expression of each of the five subunits of the core complex has been causally linked to the development or progression of human cancers. And yet, how these subunits contribute to the mechanistic roles of eIF3 remains unclear. We are investigating the mechanistic function of the eIF3 complex in translation initiation by monitoring the genome wide effects of disrupting the entire complex or portions of its mRNA-entry-channel (mEnC) arm. We have focused on two eIF3 specific mutations: eIF3a/b Degron and eIF3i DDKK. The eIF3a/b Degron mutation results in the disrupts the eIF3a and eIF3b subunits, resulting in the loss of the entire complex, whereas the eIF3i DDKK mutation results in the loss of the eIF3i and eIF3g subunits from the eIF3 mEnC arm. Using ribosome profiling of eIF3a/b Degron and eIF3i DDKK cells, we have identified specific mRNAs whose translation is most sensitive to each disruption of the eIF3 complex. The structural features and biological roles of these mRNAs shed light on the mechanistic and regulatory roles of eIF3. And yet, ribosome profiling is unable to map individual ribosome footprint reads to specific mRNA isoforms, limiting this approach. We are now conducting long-read nanopore sequencing and parallel RT-qPCR of fractionated extracts from eIF3a/b Degron and eIF3i DDKK cells. This approach will enable the identification of specific mRNA isoforms whose association with translating ribosomes is sensitive to disruption of the entire eIF3 complex or its mEnC arm, shedding new light on the roles of eIF3 and its subunits in translation initiation and its regulation. This work is supported by NIH/NIGMS R15 GM140372-01.