title: Inducing nonsense suppression by targeted pseudouridylation.
abstract: Isomerization from uridine to pseudouridine (pseudouridylation) is largely catalyzed by a family of small ribonucleoproteins called box H/ACA RNPs, each of which contains one unique small RNA-the box H/ACA RNA. The specificity of the pseudouridylation reaction is determined by the base-pairing interactions between the guide sequence of the box H/ACA RNA and the target sequence within an RNA substrate. Thus, by creating a new box H/ACA RNA harboring an artificial guide sequence that base-pairs with the substrate sequence, one can site-specifically introduce pseudouridines into virtually any RNA (e.g., mRNA, ribosomal RNA, small nuclear RNA, telomerase RNA and so on). Pseudouridylation changes the properties of a uridine residue and is likely to alter the role of its corresponding RNA in certain cellular processes, thereby enabling basic research into the effects of RNA modifications. Here we take a TRM4 reporter gene (also known as NCL1) as an example, and we present a protocol for designing a box H/ACA RNA to site-specifically pseudouridylate TRM4 mRNA. Disease-related mutation can result in early termination of translation by creating a premature termination codon (PTC); however, pseudouridylation at the PTC can suppress this translation termination (nonsense suppression). Thus, the experimental procedures described in this protocol may provide a novel way to treat PTC-related diseases. This protocol takes 10-13 d to complete.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York, USA.
authors.forename: Chao
authors.initials: C
authors.lastname: Huang
authors.forename: Guowei
authors.initials: G
authors.lastname: Wu
authors.forename: Yi-Tao
authors.initials: YT
authors.lastname: Yu
mesh_terms: D000595:Amino Acid Sequence; D018389:Codon, Nonsense; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D015316:Genetic Therapy; D008780:Methyltransferases / Q000235:genetics; D008969:Molecular Sequence Data; D011560:Pseudouridine / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D018389:Codon, Nonsense; D011560:Pseudouridine; D008780:Methyltransferases; C515610:NSUN2 protein, human