Pseudouridine RNA modification detection and quantification by RT-PCR.

Area of Science:

• Molecular Biology
• RNA Biology
• Epigenetics

Background:

• Pseudouridine (Ψ) is the most abundant RNA modification, crucial for various RNA types and dynamically regulated.
• Existing high-throughput methods for Ψ detection are not ideal for analyzing specific sites or quantitating Ψ fractions.
• A simple, quantitative method for targeted Ψ analysis is needed to investigate its functional roles.

Purpose of the Study:

• To develop a sensitive and quantitative method for analyzing specific pseudouridine (Ψ) sites and their fractions in mRNA and lncRNA.
• To enable a more convenient and rapid investigation into the function and mechanisms of Ψ modification.

Main Methods:

• The study introduces CMC-RT and ligation assisted PCR analysis of Ψ modification (CLAP), an RT-PCR and gel electrophoresis-based method.
• CLAP utilizes the N-Cyclohexyl-N'-(2-morpholinoethyl)carbodiimide (CMC) reaction, which induces reverse transcription stops at Ψ sites.
• Site-specific ligation and PCR amplification visualize Ψ-modified and unmodified RNA transcripts as distinct products on a gel.

Main Results:

• CLAP can sensitively and quantitatively assess Ψ at single-nucleotide resolution in mRNA and lncRNA.
• The method validates Ψ sites previously identified by high-throughput sequencing.
• CLAP enables the quantification of Ψ levels in specific RNA molecules.

Conclusions:

• CLAP provides a simple, sensitive, and quantitative approach for targeted pseudouridine analysis.
• This method facilitates the investigation of Ψ modification's function and mechanisms in mRNA and lncRNA.
• CLAP is a valuable tool for advancing RNA modification research.