Interactome Mapping of eIF3A in a Colon Cancer and an Immortalized Embryonic Cell Line Using Proximity-Dependent Biotin Identification

SIMPLE SUMMARY: The behavior of a cancer cell is greatly influenced by its proteome, which is the result of protein biosynthesis, modification and degradation. Eukaryotic initiation factors control protein biosynthesis and exhibit complex interactions both with each other and with the protein-coding mRNAs. Thus, the detailed molecular interactions of the eIFs might be crucial for the aggressiveness of a cancer. Here, we applied the BioID method, to analyze the interactions of eIFs using eIF3A as a prototype. As a result, we identified several known interactors of eIF3A and detected proteins not described in this context before. In particular, the occurrence of proteins involved in proper folding suggests a close coupling of protein biosynthesis and folding in cancer. This method is therefore a promising tool for further analysis of the protein biosynthesis. ABSTRACT: Translation initiation comprises complex interactions of eukaryotic initiation factor (eIF) subunits and the structural elements of the mRNAs. Translation initiation is a key process for building the cell’s proteome. It not only determines the total amount of protein synthesized but also controls the translation efficiency for individual transcripts, which is important for cancer or ageing. Thus, understanding protein interactions during translation initiation is one key that contributes to understanding how the eIF subunit composition influences translation or other pathways not yet attributed to eIFs. We applied the BioID technique to two rapidly dividing cell lines (the immortalized embryonic cell line HEK-293T and the colon carcinoma cell line HCT-166) in order to identify interacting proteins of eIF3A, a core subunit of the eukaryotic initiation factor 3 complex. We identified a total of 84 interacting proteins, with very few proteins being specific to one cell line. When protein biosynthesis was blocked by thapsigargin-induced endoplasmic reticulum (ER) stress, the interacting proteins were considerably smaller in number. In terms of gene ontology, although eIF3A interactors are mainly part of the translation machinery, protein folding and RNA binding were also found. Cells suffering from ER-stress show a few remaining interactors which are mainly ribosomal proteins or involved in RNA-binding.