Enzyme-mediated methylation and alkynylation enables transcriptome-wide identification of pseudouridine modifications

Pseudouridine (Ψ) is one of the most abundant chemical modifications and plays important roles in RNA function. Advances in our understanding of Ψ have been hindered by a limit of robust methods to precisely and sensitively map their distributions in cellular RNAs. Here, we present ELAP-seq (Enzymatic Labeling and Pull-down for Sequencing) for Ψ detection, which leverages a naturally occurring N1-methyl pseudouridine methyltransferase from Methanocaldococcus jannaschii (Mj1640). This enzyme promiscuously converts Ψ to N1-methyl-Ψ (m1Ψ) or installs a propargyl group at the same location in vitro under a mild condition, exhibiting high sensitivity and specificity, and is also functional inside cells. ELAP-seq enriches Ψ-containing RNA fragments and enables single-nucleotide-resolution Ψ detection with markedly enhanced signal-to-noise ratio and reduced sequencing and computational demands. Using ELAP-seq, we identify thousands of candidate Ψ sites in human HeLa and HEK 293T transcriptomes, validating many previously identified sites as well as reporting new ones. This versatile enzymatic platform expands the toolkit for sensitive labeling and detection of Ψ, advancing the study of RNA modification biology.