GSE175782
Identification of the stress granule transcriptome via RNA-editing in bulk S2 cells, single S2 cells and in Drosophila neurons
Stress granules are phase separated assemblies formed around mRNAs that have now been identified after stress granule purification from cell culture. Here, we present a purification free method to detect stress granules RNAs in single cells and in tissues, including those displaying cell heterogeneity. We adapted TRIBE (Target of RNA-binding proteins Identified by Editing) to detect stress-granule RNAs by fusing a stress-granule RNA-binding protein (FMR1) to the catalytic domain of an RNA-editing enzyme (ADAR). RNAs colocalized with this fusion are edited, producing mutations that are detectable by sequencing. We then show that this “in situ" method can reliably identify stress granule RNAs in single S2 cells and in Drosophila neurons, and that they encode cell cycle, transcription and splicing factors. The identification of stress granule RNAs without perturbation opens the possibility to examine cell-to-cell variability and identify the RNA content not only in stress granules, but also in other RNA based assemblies in single cells derived from tissues. Overall design: 7 samples of bulk S2 cells are analyzed, which include a non-induced sample (no expression of ADAR), 3 replicates of induced + Schneider's (basal condition) and 3 replicates of induced + arsenite (stress condition).  7 samples of Drosophila brain cells are analyzed, which include a non-induced sample (no expression of ADAR), 3 replicates of induced + Schneider's (basal condition) and 3 replicates of induced + arsenite (stress condition).  2 384-well plates are analyzed, which contain single S2 cells in each well treated with either induced + Schneider's (basal condition) or induced + arsenite (stress condition)