We report herein a novel chemical-genetic method for assaying RNA localization within living cells. RNA localization is critical for normal physiology as well as the onset of cancer and neurodegenerative disorders. Despite its importance, there is a real lack of chemical methods to directly assay RNA localization with high resolution in living cells. Our novel approach relies on in situ nucleobase oxidation by singlet oxygen generated from spatially confined fluorophores. We demonstrate that our novel method can identify RNA molecules localized within specific cellular compartments. We anticipate that this platform will provide the community with a much-needed methodology for tracking RNA localization within living cells, and set the stage for systematic large scale analysis of RNA localization in living systems.
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.
The cellular RNA pool in animals arises from two separate genomes stored in the nucleus and multiple mitochondria. Chemical methods to track nascent RNA synthesis are unable to distinguish between these two with stringency. Herein, we report that spatially restricting bioorthogonal nucleoside biosynthesis enables, for the first time, selective metabolic labeling of the RNA transcribed in the mitochondria. We envision that this approach could open the door for heretofore-impossible analyses of mitochondrial RNA. Beyond our results revealed herein, our approach provides a roadmap for researchers to begin to design strategies to examine biomolecules within subcellular compartments.
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