Abstract
Pseudouridine (Ψ) is the most common chemical modification in RNA. In eukaryotes and archaea, pseudouridine synthases, mainly guided by box H/ACA snoRNAs, convert uridine to Ψ. Ψ stabilizes RNA structure and alters RNA-RNA and RNA–protein interactions, conferring important roles in gene expression. Notably, several Ψ-linked human diseases have been identified over the years. In addition, Ψ has also been extensively used in developing mRNA vaccines. Furthermore, it has been shown that pseudouridylation can be site-specifically directed to modify specific nonsense codons, leading to nonsense suppression. All of these, together with a need to better understand the specific functions of Ψs, have motivated the development of in vitro pseudouridylation assays using purified and reconstituted box H/ACA RNPs. Here, we describe an in vitro system for box H/ACA RNA-guided RNA pseudouridylation using human cell extracts. We show that a half guide RNA (only one hairpin) is just as functionally competent as the full-length guide RNA (two hairpins) in guiding site-specific pseudouridylation in the human cell extracts. This discovery offers the opportunity for direct delivery of a short guide RNA to human cells to promote site-specific nonsense suppression and therefore has potential clinical applications.
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Acknowledgments
We thank the members of the Yu lab (past and present) for inspiring discussions during development of the method. The work performed in the Yu lab was supported by grants GM138387 and CA241111 from the US National Institutes of Health and grant CFF YU20G0 from the Cystic Fibrosis Foundation.
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Adachi, H., Chen, J.L., Yin, Q., Morais, P., Yu, YT. (2023). In Vitro Reconstitution of Pseudouridylation Catalyzed by Human Box H/ACA Ribonucleoprotein Particles. In: Lin, RJ. (eds) RNA-Protein Complexes and Interactions. Methods in Molecular Biology, vol 2666. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3191-1_14
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DOI: https://doi.org/10.1007/978-1-0716-3191-1_14
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