Promoted Read-through and Mutation Against Pseudouridine-CMC by an Evolved Reverse Transcriptase.

Area of Science:

• Molecular Biology
• RNA Biology
• Biochemistry

Background:

• Pseudouridine (Ψ) is a prevalent RNA modification crucial for RNA function and therapeutics.
• Existing methods struggle to detect Ψ at base-resolution, especially in U-rich regions where Ψ is common.

Purpose of the Study:

• To develop a novel, base-resolution method for transcriptome-wide pseudouridine mapping.
• To overcome limitations in detecting Ψ within U-rich sequences.

Main Methods:

• Utilized N-cyclohexyl N'-(2-morpholinoethyl)carbodiimide (CMC) for selective Ψ labeling.
• Employed an engineered reverse transcriptase (RT-1306) exhibiting enhanced read-through and mutation against CMC-Ψ adducts.
• Developed 'Mut-Ψ-seq' combining CMC labeling and RT-1306 for high-resolution Ψ detection.

Main Results:

• Mut-Ψ-seq successfully mapped pseudouridine at base-resolution across the transcriptome.
• The method's mutation signatures accurately identified known Ψ sites in human rRNAs.
• Identified a high-confidence list of Ψ sites in HEK-293T cell polyA-enriched RNAs using orthogonal chemical treatments.

Conclusions:

• Mut-Ψ-seq provides a robust tool for base-resolution pseudouridine mapping, particularly in challenging U-rich contexts.
• Engineered reverse transcriptases combined with selective chemical labeling offer a powerful approach for studying RNA modifications.
• This work expands the toolkit for RNA chemical modification research and provides valuable datasets for biological studies.