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

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 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 FISH to demonstrate the accumulation of multiple tRNAs in stress granules.