title: Generation and Investigation of Clinically Relevant Mutations of Translation Initiation Factor eIF3i in HEK293 Cells
Creators: name: Royce, Amelia Elizabeth
Publisher: name: University of Kent
Year: 2024

description: Messenger ribose nucleic acid (mRNA) translation initiation is an essential process involved in the control of gene expression and is the process by which ribosomes are assembled on an mRNA at the start AUG codon to allow protein synthesis to proceed. This process is facilitated by a number of translation initiation factors that in eukaryotic cells come together to help load the mRNA onto the small 40S ribosomal subunit and ultimately recruit the large 60S ribosome subunit to assemble the complete ribosome at the start codon on the mRNA ready for protein synthesis. One of these factors is called eIF3 (eukaryotic initiation factor 3) and consists in mammals of thirteen different subunits named a to m (eIF3a, eIF3b, eIF3c, etc.). eIF3i is known to be over-expressed in certain cancers and recently, six point missense mutations have been identified in connection with neurodevelopment conditions, muscular and physical deformities as well as abnormal neurology and development. These mutations are (as in the open reading frame) nucleotide 32G to A (resulting in Arg to Gln), 157G to C (Gly to Arg), 698A to G (Asn to Ser), 698A to T (Asn to Ile), 743G to A (Gly to Asp), and 772A to G (Thr to Ala). Here, investigations have been undertaken with the intent of identifying whether these mutations directly impact the function of eIF3i and/or cell phenotype using HEK293 (human embryonic kidney cells) cells as a model system. To facilitate these investigations, the wild type human eIF3i gene was initially cloned into a pcDNA3.1 plasmid, with hygromycin resistance and a V5 tag introduced at the C-terminal for easy identification using an anti-V5 antibody. The resulting cloned wild type eIF3i gene and plasmid was then subjected to site-directed mutagenesis in order to generate the six point mutations which were confirmed by Sanger sequencing. The ability to express eIF3i and the mutants was then assessed by a transient, and subsequently stable, transfection and assessment in a model HEK293 cell line and compared to appropriate controls. The mutant eIF3i proteins were all able to be expressed in HEK293 cells although not all were as well expressed as the native eIF3i suggesting that the mutation might impact expression or stability of the protein. In particular, mutations 698A to G (amino acid Asn to Ser) and 698A to T (Asn to Ile) were expressed at lower amounts compared to the other mutants and wild type suggesting that mutation at this position might be detrimental to eIF3i expression. Mutant 772A to G (Thr to Ala) was observed to be expressed at higher amounts than the other mutants and wild type. The transfected cells themselves were also studied to quantify cell culture growth, culture viability and average cell size. The total protein harvested from the cells was used to perform various western blots using the tubulin as a loading control and the V5 tag to ensure the mutants were being expressed and not just native eIF3i. Stable mutant eIF3i expressing cell lines were also blotted for eIF3a and eIF3c proteins to determine if the mutations in eIF3i affected other eIF3 subunit production. The conclusions from these experiments are that there was a difference in the ability of the cell to express some of the eIF3i mutants but not others, as indicated by lower expression than over-expressed wild type/native eIF3i. There was also, in some mutant lines a small decrease in cell size, accompanied by an increase in cell growth, possibly related to eIF3i's general oncogenic properties. There was minimal effect on cell viability or expression of the subunit eIF3a, but some variation in eIF3c expression compared to the wild type samples. In summary, this work has established systems for investigating clinically relevant mutations of eIF3i and shown that different mutations are likely differentially expressed in HEK293 cells. However, whether mutant eIF3i's can be incorporated into the eIF3 complex and associate with the other eIF3 subunits is a key question that should be investigated in future studies.