Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the...

Area of Science:

• Biochemistry
• Molecular Biology
• Genomics

Background:

• Pseudouridine (Ψ) is a prevalent RNA modification, but its functional significance is poorly understood.
• Existing detection methods lack the sensitivity and accuracy needed for comprehensive Ψ profiling.
• Accurate mapping of Ψ is crucial for understanding its role in RNA structure, function, and disease.

Purpose of the Study:

• To develop a highly sensitive and accurate method for quantitative profiling of pseudouridine (Ψ) at single-base resolution.
• To investigate the distribution and abundance of Ψ in various human RNAs and viral genomes.
• To identify targets and sequence motifs of pseudouridine synthases.

Main Methods:

• Development and application of 2-bromoacrylamide-assisted cyclization sequencing (BACS) for Ψ detection.
• BACS induces Ψ-to-C transitions, enabling quantitative mapping.
• Functional studies involving depletion of pseudouridine synthases (TRUB1, PUS7, PUS1).

Main Results:

• BACS precisely identified Ψ positions, including in densely modified regions and consecutive uridine sequences.
• All known Ψ sites in human rRNA and spliceosomal small nuclear RNAs were detected; quantitative maps generated for small nucleolar RNA and tRNA.
• BACS simultaneously detected other RNA modifications, and depletion experiments elucidated synthase targets; notably, Ψ was absent in tested viral RNAs.

Conclusions:

• BACS is a powerful tool for high-resolution, quantitative mapping of pseudouridine in RNA.
• The study provides comprehensive Ψ maps for key human RNAs and reveals insights into pseudouridine synthase functions.
• The absence of Ψ in tested viral RNAs suggests distinct RNA modification strategies across virus families.