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| Status |
Public on Feb 06, 2023 |
| Title |
Quantitative profiling of pseudouridylation landscape in the human transcriptome |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
Pseudouridine (Ψ) is an abundant post-transcriptional RNA modification in ncRNA and mRNA. However, transcriptome-wide measurement of individual Ψ sites remains unaddressed. Here, we develop “PRAISE”, via selective chemical labeling of Ψ by bisulfite to induce nucleotide deletion signature during reverse transcription, to realize quantitative assessment of the Ψ landscape in the human transcriptome. Unlike traditional RNA/DNA bisulfite treatment, our approach is based on quaternary base mapping and identifies 2,209 confident Ψ 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 Ψ 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 Ψ; we envision this approach would facilitate emerging efforts to elucidate the function and mechanism of mRNA pseudouridylation.
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| 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 Ψ 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-Ψ sites were defined by comparing the Ψ 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 Ψ 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.
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| Web link |
https://www.nature.com/articles/s41589-023-01304-7
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| Contributor(s) |
Zhang M, Jiang Z, Ma Y, Liu W, Zhuang Y, Lu B, Li K, Yi C |
| Citation(s) |
36997645 |
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| Submission date |
Aug 29, 2022 |
| Last update date |
May 08, 2023 |
| Contact name |
Jiang Zhe |
| E-mail(s) |
[email protected]
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| Phone |
010-62752895
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| Organization name |
Peking University
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| Department |
School of Life Sciences
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| Lab |
Chengqi Yi lab
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| Street address |
No. 5 Yiheyuan Road, Haidian District
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| City |
Beijing |
| State/province |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
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| Platforms (2) |
| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (38)
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| Relations |
| BioProject |
PRJNA874572 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE212210_RAW.tar |
2.2 Gb |
(http)(custom) |
TAR (of BW) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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