Promoted read-through and mutation against pseudouridine-CMC by an evolved reverse transcriptase.

Area of Science:

• Epitranscriptomics
• RNA modifications
• Molecular biology

Background:

• Pseudouridine (Ψ) is a prevalent RNA modification crucial for biological functions.
• Existing methods struggle to detect Ψ at base-resolution, particularly in U-rich sequences where Ψ is common.

Purpose of the Study:

• To develop a novel method for base-resolution mapping of pseudouridine (Ψ).
• To overcome limitations in detecting Ψ within U-rich RNA sequences.
• To expand the toolkit for epitranscriptomic studies.

Main Methods:

• Development of Mut-Ψ-seq, combining N-cyclohexyl N'-(2-morpholinoethyl)carbodiimide (CMC) chemical labeling and an engineered reverse transcriptase (RT-1306).
• CMC selectively labels Ψ sites, forming a CMC-Ψ adduct.
• RT-1306 exhibits enhanced read-through and mutation at CMC-Ψ sites, enabling detection.

Main Results:

• High-confidence pseudouridine (Ψ) sites were identified in HEK-293T cell polyA-enriched RNAs using orthogonal chemical treatments (CMC and bisulfite).
• Mutation signatures accurately resolved Ψ positions in UU-containing sequences, revealing their diverse occurrence.
• The method successfully mapped Ψ at base-resolution, including in challenging U-rich contexts.

Conclusions:

• Mut-Ψ-seq provides a robust method for base-resolution pseudouridine (Ψ) mapping.
• The technique enhances the ability to study Ψ in U-rich sequences, a significant advancement for epitranscriptomics.
• This work offers valuable methods and datasets for pseudouridine (Ψ) research and epitranscriptomic studies.