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Completed Project

Investigating the Effects of eIF3 Mutations on Start Codon Recognition

Shanya Galbokke Hewage ’23 and Professor Colin Echeverría Aitken (Biology)

Translation initiation is a complex pathway in eukaryotes and is the step by which translation is primarily regulated. The misregulation of translation initiation is a hallmark of cancer and other human diseases. Translation initiation requires the formation of the pre-initiation complex (PIC) which then attaches to the 5’ end of messenger RNA (mRNA) and then scans the mRNA in order to locate the start (AUG) codon. Translation initiation involves at least twelve different eukaryotic initiation factors. Eukaryotic initiation factor 3 (eIF3) is the largest, most complex, and least understood of these initiation factors. eIF3 participates in each phase of translation initiation and plays a large role in mRNA attachment, scanning, and start-codon recognition. In Saccharomyces cerevisiae, eIF3 is made up of 5 subunits: eIF3a, eIF3b, eIF3c, eIF3g, and eIF3i. All five of these eIF3 subunits are overexpressed in different cancer cells and are causally linked to cancer development. We combined two techniques to monitor translation in living cells. One is called ribosome profiling, which allows us to locate the position of every translating ribosome on every mRNA molecule in living cells. The other is called mRNA sequencing which is used to thoroughly determine the abundance of each individual mRNA. By comparing the information from these two approaches, we can calculate the translational efficiency of individual mRNAs and how these are impacted by mutations to eIF3. We then investigate the features of mRNAs most sensitive to specific mutations of eIF3 to infer the mechanistic contributions of eIF3 during translation initiation. I am focusing on how the presence of upstream open reading frames (uORFs) on mRNAs impact their sensitivity to mutations to eIF3. A uORF is a translatable stretch of RNA defined by a start and stop codon that is located upstream of the main coding sequence of an mRNA. uORFs force the PIC, as it is scanning, to make decisions as to whether or not it has found a start codon. Cognate uORFS begin with the universal AUG start codon, whereas near-cognate uORFs begin with a start codon that differs from AUG by one nucleotide (e.g. CUG or UUG). We observe that a mutation that disrupts the interaction of eIF3i and eIF3g with the remainder of the complex (DDKK) and a mutation that disrupts the entire eIF3 complex (eIF3 degron) both result in increased translation of near-cognate uORFs as compared to cognate uORFs. This effect is most severe on mRNAs that are sensitive to both mutations. We are currently testing these effects with statistical models. Taken together, these observations are consistent with a model in which eIF3 contributes to discrimination against near-cognate start codons, with the eIF3i and eIF3g subunits playing an important role in this discrimination