The incorporation of modified nucleotides into RNA is a powerful strategy to probe RNA structure and function. While a wide variety of modified 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 incompatibility 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 noncanonical pyrimidine nucleosides. First, 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. Next, we show that expression of mutant UCK2 in HeLa and U2OS cells is sufficient to enable the incorporation of 5-azidomethyl uridine (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.
All Science Journal Classification (ASJC) codes
- Colloid and Surface Chemistry