Quantitative profiling of RNA modifications enriched in non-membrane-bound cellular structures using APEX-RNA-MS.

The dynamic behavior of RNAs underlies fundamental biological processes. RNA function is controlled by post-transcriptional modifications that are spatiotemporally regulated, but characterizing the distribution of modified RNA transcripts with subcellular resolution is a major challenge. Here, we present APEX-RNA-MS, which combines APEX2 proximity labeling with liquid chromatography-mass spectrometry (LC-MS) quantification of modified ribonucleotides. We use APEX-RNA-MS to characterize RNA modifications proximal to RNA-binding proteins enriched in non-membrane-bound cellular structures. We measure changes in protein-proximal RNA modification levels upon induction of DNA damage foci and stress granules, consistent with previous studies using antibody-based imaging and biochemical fractionation. Further, we show that tRNA-specific modifications are proximal to G3BP1 and use RNA sequencing and RNA fluorescence in situ hybridization (FISH) to demonstrate the accumulation of multiple tRNAs in stress granules. Taken together, our work provides a general approach for characterizing the subcellular distribution of RNA modifications and reveals new insights into the composition and function of cellular condensates.