Enzyme-mediated alkynylation enables transcriptome-wide identification of pseudouridine modifications

Area of Science:

• Molecular Biology
• Biochemistry
• Genomics

Background:

• Pseudouridine (Ψ) is a prevalent RNA modification crucial for RNA function.
• Current methods for mapping Ψ distribution in cellular RNA lack sensitivity and precision.
• Understanding Ψ's role is vital for RNA biology and disease research.

Purpose of the Study:

• To develop a sensitive and precise method for mapping pseudouridine (Ψ) in cellular RNAs.
• To leverage enzymatic labeling for enhanced Ψ detection and analysis.
• To expand the toolkit for studying RNA modification biology.

Main Methods:

• Enzymatic Labeling and Pull-down for Sequencing (ELAP-seq) utilizes a methyltransferase from Methanocaldococcus jannaschii (Mj1640).
• The enzyme selectively modifies Ψ, enabling enrichment of Ψ-containing RNA fragments.
• The method achieves single-nucleotide resolution for Ψ detection with high signal-to-noise.

Main Results:

• ELAP-seq demonstrates high sensitivity and specificity for Ψ detection, functional both in vitro and in cells.
• Thousands of candidate Ψ sites were identified in human HeLa and HEK 293T transcriptomes.
• The method validated known Ψ sites and discovered novel ones, reducing sequencing and computational demands.

Conclusions:

• ELAP-seq provides a versatile and robust enzymatic platform for sensitive Ψ labeling and detection.
• This technique significantly advances the ability to map RNA modifications at single-nucleotide resolution.
• ELAP-seq expands the research toolkit for RNA modification biology and its implications.