BID-seq for transcriptome-wide quantitative sequencing of mRNA pseudouridine at base resolution

Area of Science:

• Molecular Biology
• Epigenetics
• RNA Modifications

Background:

• Pseudouridine (Ψ) is a prevalent RNA modification found in various RNA types, including mRNA.
• Functional studies of mRNA Ψ in mammals are limited by the absence of precise quantitative detection methods.
• Existing methods lack the base-pair resolution required for comprehensive Ψ mapping.

Purpose of the Study:

• To present an optimized bisulfite-induced deletion sequencing (BID-seq) protocol for quantitative mapping of Ψ distribution in cellular mRNAs.
• To enable high-resolution transcriptome-wide profiling of mRNA pseudouridylation.
• To provide a user-friendly data analysis pipeline for Ψ site detection and quantification.

Main Methods:

• Developed an optimized BID-seq protocol with streamlined library preparation and data analysis.
• Ensured high deletion ratios at Ψ sites while minimizing background deletions at unmodified uridines.
• Utilized next-generation sequencing (NGS) for transcriptome-wide Ψ profiling.

Main Results:

• The optimized BID-seq protocol is highly reproducible and sensitive, requiring as little as 10 ng of polyA+ RNA.
• Successfully identified 8,407 Ψ sites in mouse embryonic stem cells.
• The associated BID-pipe software facilitates Ψ site detection and stoichiometry quantification.

Conclusions:

• The optimized BID-seq protocol offers a robust, rapid, and quantitative method for mapping mRNA pseudouridylation.
• This workflow significantly advances the study of mRNA Ψ modifications and their functional roles in mammals.
• The protocol and analysis pipeline are accessible to researchers with basic molecular biology and bioinformatics skills.