Functional characterization of pseudouridine (Ψ) in mammalian mRNA has been hampered by the lack of a quantitative method that maps Ψ in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq), which uses a bisulfite-mediated reaction to convert pseudouridine stoichiometrically into deletion upon reverse transcription without cytosine deamination. BID-seq enables detection of abundant Ψ sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10–20 ng input RNA. We uncover consensus sequences for Ψ in mammalian mRNA and assign different ‘writer’ proteins to individual Ψ deposition. Our results reveal a transcript stabilization role of Ψ sites installed by TRUB1 in human cancer cells. We also detect the presence of Ψ within stop codons of mammalian mRNA and confirm the role of Ψ in promoting stop codon readthrough in vivo. BID-seq will enable future investigations of the roles of Ψ in diverse biological processes.

Small RNAs include tRNA, snRNA, micro-RNA, tRNA fragments and others that constitute > 90% of RNA copy numbers in a human cell and perform many essential functions. Popular small RNA-seq strategies limit the insights into coordinated small RNA response to cellular stress. Small RNA-seq also lacks multiplexing capabilities. Here, we report a multiplex small RNA-seq library preparation method (MSR-seq) to investigate cellular small RNA and mRNA response to heat shock, hydrogen peroxide, and arsenite stress. Comparing stress-induced changes of total cellular RNA and polysome-associated RNA, we identify a coordinated tRNA response that involves polysome-specific tRNA abundance and synergistic N3-methylcytosine (m3C) tRNA modification. Combining tRNA and mRNA response to stress we reveal a mechanism of stress-induced down-regulation in translational elongation. We also find that native tRNA molecules lacking several modifications are biased reservoirs for the biogenesis of tRNA fragments. Our results demonstrate the importance of simultaneous investigation of small RNAs and their modifications in response to varying biological conditions.

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.