Enhancing RNA base editing on mammalian transcripts with small nuclear RNAs

Area of Science:

• Molecular Biology
• RNA Biology
• Gene Therapy

Background:

• Endogenous uridine-rich small nuclear RNAs (U snRNAs) are crucial for pre-mRNA processing.
• Previous research established U snRNA's role in programmable exon splicing.
• The potential of snRNAs to enhance RNA base editing remained unexplored.

Purpose of the Study:

• To investigate if snRNAs can improve RNA base editing efficiency compared to existing technologies.
• To explore the application of snRNAs for targeted RNA pseudouridylation.
• To evaluate snRNA-based tools for treating genetic diseases like cystic fibrosis.

Main Methods:

• Comparison of adenosine deaminase acting on RNA (ADAR)-recruiting circular RNAs with guided A>I snRNAs for adenosine-to-inosine editing.
• Assessment of off-target gene perturbation and nuclear localization of snRNAs.
• Engineering of snRNA-H/ACA box snoRNA fusions (U>Ψ snRNAs) for targeted pseudouridylation.
• Utilizing a cystic fibrosis human bronchial epithelial cell model to assess CFTR rescue.

Main Results:

• Guided A>I snRNAs demonstrated increased adenosine-to-inosine editing efficiency, particularly for higher exon count genes.
• snRNAs showed reduced off-target gene effects and enhanced nuclear persistence compared to circular RNAs.
• A>I snRNAs efficiently edited long noncoding RNAs and pre-mRNA 3' splice sites, promoting splicing alterations.
• U>Ψ snRNAs facilitated targeted RNA pseudouridylation without DKC1 overexpression, leading to improved CFTR rescue from nonsense-mediated mRNA decay.

Conclusions:

• snRNAs represent a powerful endogenous tool for enhancing RNA base editing and pseudouridylation.
• snRNA-based approaches offer improved specificity and persistence for RNA targeting applications.
• These findings advance RNA-targeting technologies for potential therapeutic applications in genetic diseases.