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Quantitative profiling of pseudouridylation landscape in the human transcriptome.
Meiling Zhang1, Zhe Jiang1, Yichen Ma2,3
1State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.
Nature Chemical Biology
|March 30, 2023
Summary
Researchers developed PRAISE, a method to quantify pseudouridine (Ψ) RNA modifications across the human transcriptome. This technique reveals Ψ modification levels and identifies targets of key enzymes, advancing the study of RNA biology.
Area of Science:
- Molecular Biology
- Genomics
- Biochemistry
Background:
- Pseudouridine (Ψ) is a prevalent RNA modification found in both non-coding RNA (ncRNA) and messenger RNA (mRNA).
- Accurate, stoichiometric measurement of individual Ψ sites within the human transcriptome has been a significant challenge.
- Understanding the pseudouridylation landscape is crucial for deciphering RNA function and regulation.
Purpose of the Study:
- To develop and validate a novel method for quantitative assessment of the pseudouridylation landscape in the human transcriptome.
- To identify specific mRNA targets and modification stoichiometry for pseudouridine synthases.
- To quantify known and novel Ψ sites in mitochondrial mRNA.
Main Methods:
- Development of 'PRAISE' (Pseudouridine quantitative assessment via selective labeling), a method utilizing selective chemical labeling of Ψ by bisulfite.
- Induction of a nucleotide deletion signature during reverse transcription to enable quantitative measurement.
- Application of quaternary base mapping for accurate Ψ site identification and quantification.
- Perturbation of pseudouridine synthases (PUS1, PUS7, TRUB1, DKC1) to identify differential mRNA targets.
Main Results:
- The PRAISE method enabled quantitative assessment of the Ψ landscape, revealing an approximately 10% median modification level for 2,209 identified Ψ sites in HEK293T cells.
- Differential mRNA targets for PUS1, PUS7, TRUB1, and DKC1 were identified, with TRUB1 targets exhibiting the highest modification stoichiometry.
- Known and novel Ψ sites within mitochondrial mRNA, catalyzed by PUS1, were successfully quantified.
Conclusions:
- PRAISE provides a sensitive and convenient method for transcriptome-wide measurement of pseudouridine modifications.
- This quantitative approach facilitates research into the functional roles and regulatory mechanisms of mRNA pseudouridylation.
- The study significantly advances the ability to map and quantify RNA modifications, opening new avenues in RNA biology research.
