Quantification of Propargylated RNA Nucleosides After Metabolic Labeling Via the Methylation Pathway

Abstract

RNA modifications are involved in numerous biological processes and vary in different cell types. Methylation is the most widespread type of RNA modification and occurs via S-adenosyl-L-methionine (SAM). We recently developed a metabolic labeling approach based on intracellular formation of a clickable SAM analog (SeAdoYn) and demonstrated its use in mapping methyltransferase (MTase) target sites in mRNA from HeLa cells. Here we investigate how metabolic labeling via the clickable SAM analog modifies four different nucleosides in RNA of HEK293T in comparison to HeLa cells. We find that HEK293T cells retain higher cell viability upon feeding the clickable metabolic SAM precursor. In poly(A)⁺ RNA we find high A_prop/A levels (0.04 %) and in total RNA (but not poly(A)⁺ RNA) we detect prop³C, which had not been detected previously in HeLa cells. We discuss the findings in the context of data from the literature with respect to mRNA half-lives in cancer and non-cancer cell lines and suggest that CMTr2 is most likely responsible for the high A_prop level in poly(A)⁺ RNA.