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Completed Project

Dissecting the Contribution of the eif3 Subunits to Component Events During Translation Initiation

Lucy Funes ’23 and Professor Colin Echeverría Aitken (Biology)

Translation initiation is the process that assembles the ribosome, the molecular apparatus which translates the genetic code and synthesizes the corresponding protein. Assembly of the ribosome on a specific mRNA during initiation is an important step for regulating translation. Initiation begins with the assembly of a pre-initiation complex (PIC) in which the small ribosomal subunit is joined by several protein initiation factors (eIFs).  The PIC attaches to the 5’ end of an mRNA strand and scans for the start codon. Once the start codon is reached and identified, scanning stops, and the full ribosome is assembled. The largest and most complex of the eIFs is eIF3, which participates in each phase of translation initiation yet remains poorly understood. In the yeast Saccharomyces cerevisiae, eIF3 is made up of 5 subunits, identified as eIF3a, b, c, g, and i. To investigate the role of eIF3 in translation initiation, we employ ribosome profiling, which enables us to learn the position of each translating ribosome on every mRNA in living cells. We use ribosome profiling to monitor the effects of specific mutations to the eIF3 complex and investigate the features of the specific mRNAs most sensitive to these mutations. We are focusing on two mutations to eIF3: one disrupting the association of eIF3i and eIF3g with the rest of the eIF3 complex (DDKK) and another that disrupts the entire eIF3 complex (Degron). By comparing the mRNAs sensitive to both DDKK and Degron mutations to those mRNAs uniquely sensitive to the Degron mutation, we hope to disentangle the contributions of eIF3i and eIF3g from those of eIF3a, eIF3b, and eIF3c. This analysis reveals that mRNAs that appear more sensitive to disruption of eIF3i and eIF3g have longer 5’ UTR lengths and are more sensitive to mutations of the helicases Ded1 and eIF4A, or to the initiation factor eIF4B. These observations are consistent with a growing body of evidence that shows that subunits eIF3i and eIF3g play important roles in the scanning step of translation initiation.