Inducing nonsense suppression by targeted pseudouridylation.
Inducing nonsense suppression by targeted pseudouridylation.
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DOI：
10.1038/nprot.2012.029
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发表时间：
2012-03-29
期刊：
影响因子：
14.8
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尿嘧啶到伪尿嘧啶的异构化（伪尿嘧啶化）在很大程度上是由称为box H/ACA RNPs的小核糖核蛋白家族催化的，每个小核糖核蛋白家族都含有一个独特的小RNA - box H/ACA RNA。假尿嘧啶化反应的特异性是由盒状H/ACA RNA的引导序列和RNA底物内的靶序列之间的碱基配对相互作用决定的。因此，通过创建一个新的盒子H/ACA RNA，其中包含与底物序列碱基对的人工引导序列，人们可以定点地将假嘌呤引入几乎任何RNA（例如，mRNA，核糖体RNA，小核RNA，端粒酶RNA等）。假尿嘧啶化改变了尿嘧啶残基的性质，并可能改变其相应RNA在某些细胞过程中的作用，从而使RNA修饰的影响的基础研究成为可能。在这里，我们以TRM4报告基因（也称为NCL1）为例，我们提出了一种设计盒子H/ACA RNA的方案，以位点特异性地假尿嘧啶化TRM4 mRNA。疾病相关突变可通过产生过早终止密码子（PTC）导致翻译的早期终止；然而，PTC的假尿嘧啶化可以抑制这种翻译终止（无义抑制）。因此，本方案中描述的实验程序可能为治疗ptc相关疾病提供一种新的方法。该协议需要10-13天才能完成。
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.