Quantitative base-resolution sequencing technology for mapping pseudouridines in mammalian mRNA

Area of Science:

• Molecular Biology
• Epigenetics
• RNA Biology

Background:

• Posttranscriptional RNA modifications are crucial in all life forms.
• Pseudouridine (Ψ) is an abundant RNA modification in mammals, regulating diverse RNA species like rRNA, tRNA, and snRNA.
• Investigating mRNA pseudouridine (Ψ) function is challenging due to the lack of efficient transcriptome-wide mapping methods.

Purpose of the Study:

• To develop a quantitative method for transcriptome-wide mapping of pseudouridine (Ψ) in messenger RNA (mRNA).
• To enable the investigation of mRNA pseudouridine (Ψ) functions in diverse biological processes.

Main Methods:

• Developed bisulfite-induced deletion sequencing (BID-seq), a novel chemical reaction.
• Optimized bisulfite-based reaction to selectively convert pseudouridine (Ψ) into a Ψ-BS adduct without cytosine deamination.
• Utilized deletion signatures during reverse transcription to quantify Ψ modification stoichiometry at each site.

Main Results:

• BID-seq efficiently maps thousands of mRNA Ψ sites with stoichiometry information from minimal RNA input (10-20 ng polyA+ RNA).
• Identified consensus motifs for Ψ in mammalian mRNA and assigned specific 'writer' proteins to Ψ deposition.
• Confirmed the presence of Ψ within mammalian mRNA stop codons.

Conclusions:

• BID-seq provides a powerful tool for quantitative, transcriptome-wide mapping of mRNA pseudouridine (Ψ).
• This method facilitates the study of Ψ stoichiometry and its role in gene regulation.
• BID-seq lays the groundwork for future research into the functional significance of mRNA Ψ in various biological contexts.