Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using...

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• Pseudouridylation is a critical post-transcriptional modification of RNA.
• Accurate identification of pseudouridylated sites is essential for understanding RNA function and regulation.
• Existing methods often require specific chemical treatments or have limitations in throughput and resolution.

Purpose of the Study:

• To develop and apply a semi-quantitative method for high-confidence pseudouridylated site identification.
• To leverage direct long-read nanopore sequencing for RNA modification analysis.
• To uncover novel pseudouridylation sites and characterize their occupancy levels.

Main Methods:

• Direct long-read nanopore sequencing of mammalian mRNAs.
• Development of a semi-quantitative basecalling approach.
• Comparative analysis with modification-free transcriptomes to optimize parameters (coverage depth, k-mer sequences).
• Validation using synthetic RNA controls with single pseudouridine modifications.

Main Results:

• Identification of critical parameters (coverage depth, k-mer sequences) for accurate basecalling of pseudouridylated sites.
• Discovery of numerous known and previously unreported pseudouridylated sites, many located in known pseudouridine synthase target k-mers.
• Validation of identified sites, acknowledging a systematic under-calling tendency.
• Detection of mRNAs with up to 7 unique modification sites.
• Successful characterization of low-, medium-, and high-occupancy pseudouridine modifications on native RNA.

Conclusions:

• The developed method enables direct, high-confidence detection of pseudouridylation sites on native RNA using nanopore sequencing.
• The workflow facilitates the discovery of novel modification sites and characterization of their occupancy.
• This approach advances the study of RNA modifications and their biological significance.