Protocol for differential analysis of pseudouridine modifications using nanopore DRS and unmodified transcriptome...

Area of Science:

• Molecular Biology
• Genomics
• RNA Biology

Background:

• Pseudouridine is the most abundant mRNA modification.
• mRNA modifications play crucial roles in gene expression regulation.
• Dynamic changes in mRNA modifications are linked to cellular states and responses.

Purpose of the Study:

• To present a comprehensive protocol for detecting pseudouridine.
• To analyze dynamic changes in mRNA modifications using nanopore direct RNA sequencing.
• To quantify alterations in pseudouridylation across different cellular conditions.

Main Methods:

• RNA extraction and poly(A) selection.
• Nanopore direct RNA sequencing (DRS).
• In vitro transcribed (IVT) library preparation for unmodified transcriptome controls.
• Knockdown of writer proteins.
• Bioinformatic analysis including preprocessing, psi-site identification, and differential analysis.

Main Results:

• Quantification of pseudouridine sites across various cellular types and states.
• Identification of dynamic changes in mRNA modifications in response to perturbations.
• Validation of the protocol's ability to detect changes in pseudouridylation.

Conclusions:

• The presented protocol enables robust detection and analysis of pseudouridine modifications.
• This method allows for the study of dynamic changes in mRNA modifications in response to cellular perturbations.
• The findings provide a framework for investigating the functional roles of pseudouridylation in gene regulation.