Quantitative profiling of pseudouridylation landscape in the human transcriptome

Pseudouridine (?) is an abundant post-transcriptional RNA modification in ncRNA and mRNA. However, transcriptome-wide measurement of individual ? sites remains unaddressed. Here, we develop “PRAISE”, via selective chemical labeling of ? by bisulfite to induce nucleotide deletion signature during reverse transcription, to realize quantitative assessment of the ? landscape in the human transcriptome. Unlike traditional RNA/DNA bisulfite treatment, our approach is based on quaternary base mapping and identifies 2,209 confident ? sites in HEK293T cells. By perturbing pseudouridine synthases, we obtained differential mRNA targets of PUS1, PUS7, TRUB1 and DKC1. In addition, we identified known and novel ? sites in mitochondrial mRNA, which are catalyzed by a mitochondria-localized isoform of PUS1. Collectively, we provide a reliable, sensitive and convenient method to quantify transcriptome-wide ?; we envision this approach would facilitate emerging efforts to elucidate the function and mechanism of mRNA pseudouridylation. Overall design: We developed a method named "PRAISE" to detect transcriptome-wide pseudouridine sites in human mRNA and ncRNA. We first developed a tailored alignment approach and identified 18S, 28S, 5.8S rRNA sites using HEK293T total RNA (rRNA-WT_rep1,rRNA-WT_rep2,rRNA-WT_untreated), then we appiled PRAISE to characterize a quantitative ? landscape in the human nuclear-encoded and mitochondrial encoded transcriptome using HEK293T polyA+ RNA (PRAISE-WT-untreated_rep1, PRAISE-WT-untreated_rep2, PRAISE-WT_rep1,PRAISE-WT_rep2). We also employed a recently established in vitro transcribed RNA (IVT RNA) library from HEK293T transcriptome as a negative control (PRAISE-IVT-untreated, PRAISE-IVT_rep1, PRAISE-IVT_rep2). We next prepared 2nd and 3rd batches of biological replicates of HEK293T cells using the Takara v3 kit and the KAPA Strand RNA-Seq kit (PRAISE-WT-Takara-second-biological-repeats_rep1,PRAISE-WT-Takara-second-biological-repeats_rep2,PRAISE-WT-Takara-second-biological-repeats-untreated_rep1,PRAISE-WT-Takara-second-biological-repeats-untreated_rep2;PRAISE-WT-KAPA_rep1,PRAISE-WT-KAPA_rep2,PRAISE-WT-KAPA-untreated). In addition, we applied PRAISE to the cellular polyA+ RNA of three KO cell lines and one stable knockdown cell line, dependent-? sites were defined by comparing the ? profile between WT and KO (PRAISE-PUS1KO-untreated_rep1,PRAISE-PUS1KO-untreated_rep2, PRAISE-PUS1KO_rep1,PRAISE-PUS1KO_rep2,PRAISE-DKC1KD_rep1,PRAISE-DKC1KD_rep2,PRAISE-DKC1KD-untreated_rep1,PRAISE-DKC1NC_rep1,PRAISE-DKC1NC_rep2,PRAISE-DKC1NC-untreated_rep1,PRAISE-PUS7KO-untreated_rep1,PRAISE-PUS7KO-untreated_rep2, PRAISE-PUS7KO_rep1,PRAISE-PUS7KO_rep2,PRAISE-TRUB1KO-untreated_rep1,PRAISE-TRUB1KO-untreated_rep2, PRAISE-TRUB1KO_rep1,PRAISE-TRUB1KO_rep2). We also applied the small RNA fraction of WT HEK293T to identify mitochondrial tRNA ? sites (PRAISE-WT-tRNA_rep1,PRAISE-WT-tRNA_rep2,PRAISE-WT-tRNA-untreated). Please note that the GSM6513552, GSM7029102 records have been updated on Mar 25, 2023.