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

Functional characterization of pseudouridine (?) in mammalian messenger RNA (mRNA) has been hampered by the lack of a quantitative method that maps pseudouridine in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq) that utilizes a bisulfite-mediated reaction to stoichiometrically convert pseudouridine into deletion upon reverse transcription. BID-seq enabled detection of abundant pseudouridine sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10-20 ng input RNA. We uncovered consensus sequences for pseudouridine in mammalian mRNA and assigned different 'writer' proteins to individual pseudouridine deposition. Our results reveal a transcript stabilization role of ? sites installed by TRUB1 in human cancer cells. We also detected presence of ? within stop codons of mammalian mRNA, and confirmed the role of ? in promoting stop codon read-through in vivo. This new method for sensitive and comprehensive detection of ? sets the stage for future investigations of the roles of ? in diverse biological processes. Overall design: [dataset1] 20 samples (Sample 1-20): "Input" and "Treated" samples in BID-seq to detect pseudouridine in polyA+ RNA from HeLa cells of shControl and 8 human PUS synthase knockdown. (5' SR Adapter: 5'-GUUCAGAGUUCUACAGUCCGACGAUC NNNNN-3'; See instructions of each sample about 3' SR Adapter sequences) [dataset3] 48 samples (Sample 41-88): "Input" and "Treated" samples in BID-seq to detect pseudouridine in polyA+ RNA from 12 mouse tissues. (5' SR Adapter: 5'-GUUCAGAGUUCUACAGUCCGACGAUC NNNNN-3'; See instructions of each sample about 3' SR Adapter sequences) [dataset4] 18 samples (Sample 89-106): "Input" and "Treated" samples in BID-seq to detect pseudouridine in polyA+ RNA from wild-type HeLa, HEK293T and A549 cells. (5' SR Adapter: 5'-GUUCAGAGUUCUACAGUCCGACGAUC-3'; See instructions of each sample about 3' SR Adapter sequences)