Updated Pseudo-seq Protocol for Transcriptome-Wide Detection of Pseudouridines

Area of Science:

• Molecular Biology
• Genomics
• RNA Biology

Background:

• Pseudouridine (Ψ) is the most abundant modification in cellular RNAs, found in various RNA types including messenger RNAs (mRNAs).
• Existing methods for Ψ identification face limitations with current sequencing technologies due to reagent incompatibility.
• Accurate mapping of pseudouridylation sites is crucial for understanding RNA regulation and function.

Purpose of the Study:

• To present an updated Pseudo-seq technique for genome-wide identification of pseudouridylation sites with single-nucleotide precision.
• To provide a comprehensive protocol for Pseudo-seq adaptable to diverse cell and tissue types.
• To enable discovery of novel pseudouridylation sites and facilitate functional studies.

Main Methods:

• Development and description of an updated Pseudo-seq protocol.
• Includes optimized RNA isolation from human cells, efficient library preparation using current materials, and detailed data analysis.
• Methodology is designed for high-quality mRNA isolation and compatibility with modern sequencing platforms.

Main Results:

• The updated Pseudo-seq method accurately identifies pseudouridine (Ψ) sites on mRNAs.
• Provides precise positional and quantitative information regarding Ψ modifications.
• Demonstrates efficient library preparation compatible with the latest sequencing technologies.

Conclusions:

• The updated Pseudo-seq is a valuable tool for genome-wide pseudouridylation site identification.
• The methodology is adaptable and facilitates the discovery of novel Ψ sites in various biological contexts.
• This technique is essential for advancing the understanding of pseudouridine's role in RNA regulation and function.