Targeting APEX2 to the mRNA encoding fatty acid synthase β in yeast identifies proteins that bind and control its translational efficiency in the cell cycle

Abstract

Profiling the repertoire of proteins associated with a given mRNA during the cell cycle is unstudied. Furthermore, it is much easier to ask and answer what mRNAs a specific protein might bind to than the other way around. Here, we implemented an RNA-centric proximity labeling technology at different points in the cell cycle in highly synchronous yeast cultures. To understand how the translation of FAS1, encoding fatty acid synthase, peaks late in the cell cycle, we identified proteins that bind the FAS1 transcript in a cell cycle-dependent manner. We used dCas13d-APEX2 fusions to target FAS1 and label nearby proteins, which were then identified by mass spectrometry. The glycolytic enzyme Tdh3p, a known RNA-binding protein, bound the FAS1 mRNA, and it was necessary for the increased Fas1p expression late in the cell cycle. Lastly, cells lacking Tdh3p had altered size homeostasis, consistent with delayed G1/S transition and exit from mitosis. These results point to unexpected connections between major metabolic pathways. They also underscore the role of mRNA-protein interactions for gene expression during cell division.