Mapping the interactome of human tRNA methyltransferase TRMT1 using dual proximity labeling

Abstract

Transfer RNA methyltransferase 1 (TRMT1) installs N2-methylguanosine and N2,N2-dimethylguanosine modifications at position 26 of mammalian tRNAs, supporting tRNA structure, translation, and cellular response to redox stress. However, the local environment and interactome of TRMT1 in the cell is poorly defined. Here, we use APEX2-based proximity labeling of the N- and C-terminus of TRMT1, coupled with label-free quantitative proteomics to map candidate TRMT1-proximal proteins in HEK293T cells. Mass spectrometry data was acquired using both data-independent acquisition (DIA) and data-dependent acquisition (DDA) methods, and it was found that DIA substantially increased proximity proteome coverage, reproducibility, and the number of significantly enriched candidate hits compared to the DDA method. N- and C-terminal APEX2-TRMT1 constructs captured largely overlapping proteomes, suggesting the dual-labeling strategy provides a robust map of proximal proteins. Analysis of the significant TRMT1-proximal proteins reveals enrichment in RNA processing and ribonucleoprotein-associated factors, in addition to hits connected to tRNA modification, tRNA biogenesis, and redox-associated biology. These data provide a proteome-scale view of TRMT1-associated cellular proteins and environments, and lay the groundwork for future validation of functional TRMT1 interaction networks. SignificanceO_LIFusing APEX2 enzyme to both N-terminal and C-terminal of the bait enhanced the sensitivity for identification of protein interactions. C_LIO_LICombining APEX2-based endogenous labeling with DIA mass spectrometry increases reproducibility and depth of proximity proteome. C_LIO_LIThe study provides a rich source of potential interacting or proximally close proteins to TRMT1, which warrants further validation studies. C_LI