Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome

Pseudouridine (?) is one of the most abundant modifications in cellular RNA. However, its function remains elusive, mainly due to the lack of highly sensitive and accurate detection methods. To address this challenge, we introduced 2-bromoacrylamide-assisted cyclization sequencing (BACS) for quantitative profiling of ? at single-base resolution. Based on novel bromoacrylamide cyclization chemistry, BACS enables a ?-to-C transition. Compared to previous methods, BACS allowed the precise identification of ? positions, especially in densely modified ? regions and consecutive uridine sequences. BACS successfully detected all known ? sites in human rRNA and spliceosomal snRNAs and generated the first quantitative ? map of human snoRNA and tRNA. Furthermore, BACS simultaneously detected adenosine-to-inosine (A-to-I) editing sites and N1-methyladenosine (m1A). Depletion of three key pseudouridine synthases (PUS) enabled us to elucidate the targets and sequence motifs of TRUB1, PUS7, and PUS1 in HeLa cells. We further applied BACS to Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) and identified a highly abundant ?114 site in EBER2. Surprisingly, applying BACS to a panel of RNA viruses demonstrated the absence of ? in their viral transcripts or genomes, shedding light on differences in pseudouridylation between virus families. We anticipate BACS to serve as a powerful tool to uncover the biological importance of ? in future studies. Overall design: Dataset1: 8 total RNA samples from HeLa. Sample1-8 corresponds to samples containning different fractions of NN?NN spike-in.(0%,20%,50%,75%, respectively).Dataset2(sample 9-12):4 Ribo-depleted library for ncRNAs. Dataset3(sample 13-18): 4 PolyA+ RNA samples from HeLa and 4 for IVT samples. Dataset4(sample 19-32): Ribo-depleted libraries for PUS7,PUS1 and TRUB1 knockout cell lines in Hela. Dataset5(sample 33-50): Ribo-depleted libraries for Epstein–Barr virus(EBV), SARS-CoV-2, Hepatitis C(HCV),Zika, Hepatitis Delta Virus and Sindbis virus samples. Ribo-depleted libraries for Epstein-Barr virus(EBV) using modified BID-seq (Samples 51-54).