Pseudouridine in mRNA: Incorporation, Detection, and Recoding.
Pseudouridine in mRNA: Incorporation, Detection, and Recoding.
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DOI：
10.1016/bs.mie.2015.03.009
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发表时间：
2015
影响因子：
--
通讯作者：
Yu YT
中科院分区：
文献类型：
--
作者：
Wu G;Huang C;Yu YT
人们早就知道假尿嘧啶（Ψ）是稳定rna（包括tRNA、rRNA和snRNA）中最丰富的修饰核苷酸。最近使用大规模平行测序的研究发现，mrna中也存在数百个Ψs。在真核生物和古细菌中，RNA假尿嘧啶化主要由盒状H/ACA RNPs引入，RNA -蛋白复合物由单个RNA片段和四个核心蛋白组成。已经确定Ψ在几种模式生物（包括酵母、非洲爪蟾和人类）中对稳定rna的结构和功能的调节起着至关重要的作用。然而，Ψ在mRNA中的功能作用仍有待阐明。一种可能性（对于停止/终止密码子也是如此）是Ψ在翻译过程中影响解码。因此，有必要建立一种系统，将假尿嘧啶化位点特异性地引入到靶mRNA中，并生化检测mRNA假尿嘧啶化对蛋白质翻译的影响。在这里，我们提出了一种方法：(1)通过设计盒H/ACA RNP将尿苷位点特异性转化为mRNA中的Ψ，(2)使用位点特异性标记，然后进行核酸酶切和薄层色谱检测目标mRNA中的Ψ，以及(3)分析翻译过程中假尿苷化过早终止密码子的重编码。
It has long been known that pseudouridine (Ψ) is the most abundant modified nucleotide in stable RNAs, including tRNA, rRNA, and snRNA. Recent studies using massive parallel sequencing have uncovered the presence of hundreds of Ψs in mRNAs as well. In eukaryotes and archaea, RNA pseudouridylation is introduced predominantly by box H/ACA RNPs, RNA–protein complexes each consisting of a single RNA moiety and four core proteins. It has been well established that Ψ plays an essential role in regulating the structure and function of stable RNAs in several model organisms, including yeast, Xenopus laevis, and humans. However, the functional role of Ψ in mRNA remains to be elucidated. One possibility (and true for stop/termination codons) is that Ψ influences decoding during translation. It is imperative, therefore, to establish a system, in which one can site-specifically introduce pseudouridylation into target mRNA and biochemically test the impact of mRNA pseudouridylation on protein translation. Here, we present a method for (1) site-specific conversion of uridine into Ψ in mRNA by designer box H/ACA RNP, (2) detection of Ψ in target mRNA using site-specific labeling followed by nuclease digestion and thin layer chromatography, and (3) analysis of recoding of pseudouridylated premature termination codon in mRNA during translation.
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影响因子：
64.8
作者：
Fernandez, Israel S.;Chyan Leong Ng;Kelley, Ann C.;Wu, Guowei;Yu, Yi-Tao;Ramakrishnan, V.
通讯作者：
Ramakrishnan, V.
影响因子：
7.3
作者：
Wu, Guowei;Yu, Andrew T.;Kantartzis, Athena;Yu, Yi-Tao
通讯作者：
Yu, Yi-Tao
RNA 假尿苷化：对旧修饰的新见解。
影响因子：
13.8
作者：
Ge, Junhui;Yu, Yi-Tao
通讯作者：
Yu, Yi-Tao
影响因子：
3.7
作者：
Lovejoy AF;Riordan DP;Brown PO
通讯作者：
Brown PO
DOI：
10.1007/978-1-61779-018-8_14
发表时间：
2011
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Huang, Chao;Karijolich, John;Yu, Yi-Tao
通讯作者：
Yu, Yi-Tao