Eukaryotic mRNA translation is a complicated and highly controlled process involving the formation of many complexes and association of ‘helper’ molecules. It begins with initiator transfer RNA (MettRNAi), guanosine triphosphate (GTP) and eukaryotic initiation factor 2 (eIF2) forming a ternary complex and secondly recruiting eIF3, eIF1 and eIF1A to form the 43S preinitiation complex (PIC). The largest and most complex molecule to play a role in this process is eIF3, composed of 13 protein subunits. eIF3 is involved in the controlled scanning of mRNA for start codons when forming the 43S preinitiation complex with other eIFs and ribosomal 40S. eIF3i is a beta-propeller structure containing seven WD40 domain repeats and considered an interactive scaffold structure for protein-protein interactions. eIF3i has many non-canonical associations such as in growth and proliferation, angiogenesis, anchorage-independent growth, AKT signalling, COX-2 synthesis and Transforming growth factor β signalling. Six clinically relevant mutations have been identified as responsible for pathogenesis in neurodevelopment, all comprising of a point mutation in the eIF3i sequence that results in a missense mutation in the amino acid sequence (M1: Arg11Gln (32G>A), M2: Thr258Ala (772A>G), M3: Asn233Ser (698A>T), M4: Gly248Asp (743G>D), M5: Gly53Arg (157G>C), M6: Asn233Ile (698A>T). The aims of this study were to start investigating the implication of the 6 mutations of eIF3i on its function, impact on cell phenotype, and gain insights into their potential role in disease pathogenesis and any link to TGFBR2 interaction. Restriction enzyme traditional cloning and Sequence and ligation independent cloning (SLIC), was used to introduce the 6 eIF3i mutations and the eIF3i wild type into pcDNA5/FRT vector tagged with a C-terminal V5 sequence. Flp-In-293 HEK cells were then co-transfected with pOG44 and the constructed mutant eIF3i vectors to generate stable, site specific integrated eIF3i mutant expressing cell lines. eIF3i mutant and wild type cell lines were characterized by taking samples at batch culture day 2, 4, 7 and 9 and analysing these using western blot and RT-qPCR. The stable cell lines were characterized for eIF3i and TGFBR2 relative expression. Immunofluorescent microscopy on transiently transfected Flp-In-293 HEK cells and in-silico methods were also used to further analyse eIF3i and TGFBR2 interactions. The results of the cell culture growth study showed that M1(32G>A), M2 (772A>G) and M5 (157G>C) may act as hypomorphs, exhibiting less than normal gene function, with no change in growth kinetics as compared to the wild type. AlphaFold structural modelling analysis of the docking interface of TGFBR2:eIF3i found that M1 (32G>A), M2 (772A>G) and M5 (157G>C), localized on the interface and may explain M1 (32G>A) expressing at a higher amount compared to TGFBR2 than the other variants due to it localizing at the core of the interface (A, M2: 772A>G, and M5: 157G>C) exhibit hypomorphic behaviour, leading to altered cell growth patterns and potentially changes in TGFBR2 expression. More research is needed to validate these findings in vivo and to explore their broader implications for eIF3i-mediated TGF-β signalling and disease pathogenesis.
Monika Petraviciute (Mon,) studied this question.