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

Abstract

Pseudouridine (Ψ) is one of the most abundant chemical modifications that exists in various types of RNA species and is known to play important roles in RNA function. The advances in studies of Ψ in less abundant messenger RNA species have been hindered by a lack of suitable methods to precisely and sensitively map their distributions. Here we show that a methyltransferase from Methanocaldococcus jannaschii can label RNA Ψ efficiently and specifically with various functional groups, both in isolated RNA and inside cells. We leveraged this enzymatic labeling strategy to develop ELAP-seq as a facile method to enrich Ψ-modified transcripts for the detection of Ψ modifications at single base resolution with high sensitivity and low background. Using this method, we identified over 10, 000 candidate Ψ sites from human transcripts, which provides new insights into Ψ biosynthesis and function. Our study provides a chemical biology method that specifically labels Ψ for its detection and functional alteration.

Bisulfite and Nanopore Sequencing for Pseudouridine in RNA

“…The ultimate goal of finding an efficient method to biotinylated Ψ specifically using a suitably functionalized nucleophile for addition to C6 of the pyrimidine or for trapping of intermediates 3 and 4 has not yet been successful. An alternative was recently presented by the He laboratory who used metabolic labeling to propargylate N1 of Ψ . This can then be used to pull down low occupancy sites in RNA fragments for sequencing.…”