PUS7 mRNA pseudouridylation is driven by RNA sequence, structure, and cell-type-specific features

Area of Science:

• Molecular Biology
• Epitranscriptomics
• RNA Modification

Background:

• PUS7 is a key mRNA pseudouridine synthase implicated in neurodevelopmental disorders and cancer.
• The specificity mechanisms of PUS7, despite its recognition of a degenerate UNUAR sequence, are not well understood.

Purpose of the Study:

• To investigate the regulatory features and specificity determinants of PUS7.
• To develop a method for high-throughput pseudouridine detection to study PUS7 activity.
• To understand how PUS7 activity is regulated in cellular contexts.

Main Methods:

• Development of Nano-Mod-Amp, a targeted Nanopore high-throughput method for pseudouridine detection.
• Analysis of PUS7 modification patterns based on RNA sequence, uridine accessibility, and RNA structure.
• Experimental perturbation of RNA structure using mutations and antisense oligonucleotides.
• Correlation of pseudouridine levels with PUS7 expression and activity across different cell types and states.

Main Results:

• Identified USUAG sequence, target uridine accessibility, and RNA structure as critical drivers of PUS7-mediated mRNA modification.
• Demonstrated that modulating RNA structure affects pseudouridine levels.
• Showed that pseudouridine levels correlate with PUS7 levels in cells, indicating regulatory potential.
• Observed variations in PUS7 activity across cell types independent of expression levels, suggesting roles for other factors.

Conclusions:

• Elucidated key principles governing PUS7 activity and specificity.
• Enabled site-specific modulation of pseudouridines, providing tools for epitranscriptomic research.
• Offered molecular insights into the regulation and dysregulation of PUS7 in disease contexts.