Pseudouridine site assignment by high-throughput in vitro RNA pseudouridylation and sequencing

Area of Science:

• Molecular Biology
• RNA Biology
• Biochemistry

Background:

• Pseudouridine (Ψ) is the most abundant RNA modification, influencing RNA structure, protein interactions, and gene expression.
• While many pseudouridine synthases (PUS) exist, their specific RNA targets and modification determinants are often unknown.
• Understanding PUS specificity is crucial for elucidating the functional roles of Ψ in mRNA metabolism.

Purpose of the Study:

• To develop and validate a high-throughput in vitro assay for pseudouridylation.
• To identify direct RNA targets of pseudouridine synthases (PUS).
• To investigate RNA sequence and structural features governing PUS specificity.

Main Methods:

• Development of a high-throughput in vitro pseudouridylation assay coupled with sequencing.
• Implementation of an analysis pipeline for assigning Ψ sites, including improved peak-calling for low-abundance transcripts.
• Validation of candidate pseudouridine sites identified in cellular contexts.

Main Results:

• The assay successfully validates candidate pseudouridine sites and assigns them as direct PUS targets.
• Identification of RNA sequence and structural determinants that direct pseudouridylation.
• The analysis pipeline effectively handles noisy data from low-abundance transcripts.

Conclusions:

• The developed assay provides a powerful tool for mapping pseudouridylation sites and understanding PUS specificity.
• This work facilitates the study of Ψ's impact on mRNA metabolism and gene expression.
• The findings pave the way for deeper investigations into the functional significance of specific pseudouridine modifications.