Identification of proteins interacting with translating RPAC reporter mRNAs (FGH17)

To discover new regulators of Regulatory Particle Assembly Chaperone (RPAC) translation, we identified RNA-binding proteins (RBPs) with increased recruitment to translating WT FGH17 mRNAs compared to non-translatable FGH17-40ntΔ mRNAs in vivo. To this end, we first treated yeast cells with rapamycin to stimulate translation of FGH17 mRNAs. Ribosomes were locked on mRNAs using cycloheximide and UV-crosslinking covalently linked the RNA and any bound proteins together. We next used anti-FLAG beads to immunoprecipitate translated FGH17 protein, as well as any translating FGH17-mRNA complexes where locked ribosomes had already synthesized one or both N-terminal FLAG tags. As FGH17-40ntΔ mRNAs are not translated, these samples should only immunoprecipitate proteins that bind non-specifically to the anti-FLAG beads. Based on the prediction that potential regulators of RPAC translation would be UV crosslinked to FGH17 mRNA within the 5’ UTR and upstream of locked translating ribosomes, we used RNases to specifically elute these potential regulators. We then identified the proteins in the RNase elution by tandem mass tag (TMT)-based quantitative proteomics.