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The incorporation of chemically modified nucleotides into RNA is a powerful strategy to probe RNA function and has been exploited in the development of anti-cancer and anti-viral drugs. While a wide variety of mod-ified nucleotides can be incorporated into RNA in vitro using chemical or enzymatic synthesis, strategies for the metabolic incorporation of artificial nucleotides into cellular RNA are limited, largely due to the incompati-bility of modified nucleobases and nucleosides with nucleotide salvage pathways. In this work, we develop a metabolic engineering strategy to facilitate the labeling of cellular RNA with non-canonical pyrimidine nucleosides. We use structure-based protein engineering to alter the substrate specificity of uridine-cytidine kinase 2 (UCK2), a key enzyme in the pyrimidine nucleotide salvage pathway. First, we show in biochemical assays with purified protein that a single amino acid substitu-tion in the UCK2 active site promotes phosphorylation of 5-azidomethyluridine (5-AmU), a uridine analog that is not processed by endogenous nucleotide salvage pathways. Further, expression of mutant UCK2 in HeLa cells is sufficient to enable the incorporation of 5-AmU into cellular RNA and promotes RNA labeling by other C5-modified pyrimidines. Finally, we apply UCK2-mediated RNA labeling with 5-AmU to study RNA trafficking and turnover during normal and stress conditions and find diminished RNA localization in the cytosol during arsenite stress. Taken together, our study provides a general strategy for the incorporation of modified pyrimidine nucleosides into cellular RNA and expands the chemical toolkit of modified bases for studying dynamic RNA behavior in living cells.
PMID: 30735369 [PubMed - as supplied by publisher]