Pseudouridine Detection and Quantification Using Bisulfite Incorporation Hindered Ligation.

Area of Science:

• Molecular Biology
• RNA Biology
• Biochemistry

Background:

• Pseudouridine (Ψ) is a crucial RNA modification found across diverse RNA types, including mRNA and lncRNA.
• Accurate detection and quantification of Ψ at single-nucleotide resolution are essential for understanding its functional roles.
• Existing methods, like CMC labeling, suffer from RNA degradation, high input requirements, and time-consuming procedures.

Purpose of the Study:

• To develop a robust and efficient method for detecting and quantifying pseudouridine (Ψ) modifications in various RNA species.
• To overcome the limitations of RNA degradation and complexity associated with previous Ψ detection techniques.
• To enable high-throughput and quantitative analysis of Ψ dynamics in RNA.

Main Methods:

• Introduction of the Bisulfite Incorporation Hindered ligation-based method (BIHIND) for Ψ detection.
• Coupling BIHIND with quantitative PCR (BIHIND-qPCR) for site-specific Ψ quantification.
• Integration of BIHIND with next-generation sequencing (BIHIND-seq) for high-throughput Ψ profiling without reverse transcription.

Main Results:

• BIHIND successfully detects and quantifies Ψ modifications in rRNA, mRNA, and noncoding RNA.
• BIHIND-qPCR provides quantitative fractions of Ψ at individual modification sites.
• BIHIND-seq enables high-throughput Ψ sequencing, offering a significant advancement over prior methods.
• The method's robustness was confirmed by analyzing Ψ dynamics after pseudouridine synthase depletion.

Conclusions:

• The BIHIND method offers a sensitive, efficient, and versatile approach for Ψ detection and quantification across multiple RNA types.
• BIHIND overcomes the drawbacks of previous methods, reducing RNA degradation and simplifying workflows.
• This technique facilitates deeper investigation into the functional significance of pseudouridylation in RNA biology.