Investigating the Functional Impact of Six Clinically Relevant Variants in Eukaryotic Translation Initiation Factor 3 subunit i (eIF3i) on Global Translation, Growth Rate, and Protein Interaction: A Molecular and Cellular Analysis

mRNA translation is a critical control point in gene expression and biological processes. mRNA translation allows cells and organisms to alter the expression of different genes, at the protein level, according to their needs. The process of mRNA translation is thus highly regulated and complex, involving multiple complex protein interactions and rearrangements. Translation initiation, one of four key stages of translation, is the main focus of this research project. In eukaryotes mRNA translation initiation involves the recruitment of initiation factors, charged tRNA, the 40S ribosomal subunit and the 60S ribosomal subunit, to an activated looped mRNA transcript to allow the formation of the 80S ribosome complex on the mRNA, posed and ready for the next stage of translation, elongation. One of many essential translation initiation factors involved in the translation initiation process in mammalian cells is eukaryotic translation initiation factor 3 subunit i (eIF3i), a component of the 13-subunit eIF3 complex. eIF3i, encoded by the EIF3I gene, is highly conserved and ubiquitously expressed across mammalian tissues. Notably, eIF3i is considered a proto-oncogene, as research has shown it to be overexpressed in certain cancers, where it promotes tumorigenesis and cell proliferation. Overexpression of eIF3i has been linked to colon, breast, head, and neck cancers. Unpublished data from collaborators in Italy have identified six clinically relevant point mutations in eIF3i, which are associated with neurodevelopmental impairments, behavioural abnormalities, and skeletal defects. This project investigated these six point mutations, each resulting in a single amino acid change, by transfecting C-terminal V5-tagged eIF3i plasmids (either wildtype or one of the mutated forms) into HEK293 cells to assess the functional impact of these de novo mutations. The study evaluated several characteristics, comparing the effects of mutant eIF3i expression with wildtype eIF3i overexpression. Protein-protein interactions of eIF3i were examined using immunoprecipitation assays, while global translation activity was assessed through nano-luciferase assays. Additionally, the effects of mutant eIF3i on culture viability, cell growth, and cell diameter were studied using a Vi-Cell instrument and an real-time cell analysis (RTCA) dual purpose (DP) instrument. Key findings from the assays showed that cells expressing mutated eIF3i exhibited faster growth and achieved a higher maximal cell index compared to cells overexpressing wild type eIF3i. Two specific mutants designated Mutant 5 and Mutant 6, produced cell populations with the highest growth rates and cell indices. These results suggest that the mutations confer a growth advantage to the cells. However, despite the increased growth rates, the global translation activity in cells expressing mutant eIF3i was found to be reduced in pools expressing eIF3i transiently but not stably. Immunoprecipitation assays revealed that despite the presence of the mutations, eIF3i was still able to interact with other components of the eIF3 complex (e.g., eIF4G, eIF3c, eIF3j, eIF3a, and eIF3b). Importantly, the mutations did not appear to affect cell viability or culture diameter. In conclusion, this research underscores the need for further investigation into how eIF3i mutations contribute to disease phenotypes, potentially involving roles for eIF3i outside the eIF3 complex. Moreover, further studies are recommended to understand the mechanisms by which mutated eIF3i enhances cell growth while decreasing global translation activity under certain conditions, and how these findings may relate to the neurodevelopmental disorders observed in patients with mutant eIF3i.