Project description:The dynamic behavior of RNA 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 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. 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.
Project description:To address specificity of ALaP-seq for PMLwt, we performed genome-wide profiling of genomic regions associated with NLS-APEX or PMLca-APEX. Cells were treated with H2O2 to trigger labeling of chromatin with biotin (H2O2+). Experimental negative control for PMLca (H2O2-), where the H2O2 treatment was omitted, was also analyzed.
Project description:Stress granules are dynamic non-membrane bound organelles made up of untranslating messenger ribonucleoproteins (mRNPs) that form when cells integrate stressful environmental cues resulting in stalled translation initiation complexes. Although stress granules dramatically alter mRNA and protein localization, understanding these complexes has proven to be challenging through conventional imaging, purification, and crosslinking approaches. We therefore developed an RNA proximity labeling technique, APEX-Seq, which uses the ascorbate peroxidase APEX2 to probe the spatial organization of the transcriptome. We show that APEX-Seq can resolve the localization of RNAs within the cell and determine their enrichment or depletion near key RNA-binding proteins. Matching both the spatial transcriptome using APEX-seq, and the spatial proteome using APEX-mass spectrometry (APEX-MS) provide new insights into the organization of translation initiation complexes on active mRNAs, as well as revealing unanticipated complexity in stress granule contents, and provides a powerful approach to explore the spatial environment of macromolecules.