Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution

Abstract

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.

Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct and long-read sequencing

“…Treatment with a strong base removes the CMC from all the sites except for the N3 position of ψ. Recently, the use of an RNA bisulfite reaction was demonstrated for ψ-specific labeling 23,24 . Chemical labeling of ψ combined with next-generation sequencing 3,18,23 has yielded over 2,000 putative ψ sites within mammalian mRNAs, but different methods identified different sites with limited overlap 25 , pointing to a need for alternative detection methods.…”

BID-seq: The Quantitative and Base-Resolution Sequencing Method for RNA Pseudouridine

“…BID-seq detected several thousand Ψ sites with modification fraction information in three human cell lines and 12 mouse tissues. 7 The quantitative ability of BID-seq facilitated us to monitor Ψ stoichiometry changes and assigned different "writer" proteins to individual Ψ sites in mRNA. For some mRNAs that contain a highly modified Ψ within the GUΨC motif, we demonstrated the functional role of TRUB1 as the main Ψ "writer" protein that seems to regulate mRNA stability through Ψ deposition.…”

“…Furthermore, BID-seq confirms the presence of Ψ within mammalian mRNA stop codons, which promotes stop codon read-through in vivo. 7 BID-seq has four key advantages over CMC-based approaches: [4][5][6]18 (1) Deletion signatures are rare in naturally transcribed rRNA, mRNA, tRNA, and other noncoding RNAs, giving very low background noise in BID-seq. After BID-seq treatment, the newly generated deletion signatures can be detected by bioinformatic analysis, which is more distinct than RT truncation.…”

“…This unique deletion profile at each Ψ-modified site set the basis for modification stoichiometry quantification in a more clean and accurate way (Table 1). 7 (2) BID-seq does not require pulldown enrichment and can determine Ψ modification stoichiometry at each modified site. (3) BIDseq starts with ∼10 ng of polyA + RNA from cultured cells or animal tissues (Table 1), 7 which corresponds to ∼500 ng of cellular total RNA or RNA isolated from ∼20,000 cells.…”

“…7 (2) BID-seq does not require pulldown enrichment and can determine Ψ modification stoichiometry at each modified site. (3) BIDseq starts with ∼10 ng of polyA + RNA from cultured cells or animal tissues (Table 1), 7 which corresponds to ∼500 ng of cellular total RNA or RNA isolated from ∼20,000 cells. The mild treatment in BID-seq bisulfite avoids RNA decay and enables the use of ∼10 ng of RNA input in sequencing library preparation, making it compatible with diverse biological sample types.…”