Chapter title |
In Vitro Reconstitution of Pseudouridylation Catalyzed by Human Box H/ACA Ribonucleoprotein Particles.
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Chapter number | 14 |
Book title |
RNA-Protein Complexes and Interactions
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Published in |
Methods in molecular biology, January 2023
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DOI | 10.1007/978-1-0716-3191-1_14 |
Pubmed ID | |
Book ISBNs |
978-1-07-163190-4, 978-1-07-163191-1
|
Authors |
Adachi, Hironori, Chen, Jonathan L, Yin, Qiangzong, Morais, Pedro, Yu, Yi-Tao, Chen, Jonathan L. |
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. |
X Demographics
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United States | 1 | 25% |
Unknown | 3 | 75% |
Demographic breakdown
Type | Count | As % |
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Members of the public | 4 | 100% |