Improved Analysis of RNA Localization by Spatially Restricted Oxidation of RNA–Protein Complexes

Abstract

Recent analysis of transcriptomes has revealed that RNAs perform a myriad of functions beyond encoding proteins. Critical to RNA function is its transport to unique subcellular locations. Despite the importance of RNA localization, it is still very challenging to study in an unbiased manner. We recently described the ability to tag RNA molecules within subcellular locations through spatially restricted nucleobase oxidation. Herein, we describe a dramatic improvement of this protocol through the localized oxidation and tagging of proteins. Isolation of RNA-protein complexes enabled the enrichment of challenging RNA targets on chromatin and presented a considerably optimized protocol for the analysis of RNA subcellular localization within living cells.