NONSENSE RNA SURVEILLANCE IN HEALTH AND DISEASE

描述(改编自应用)：所有生物从真细菌到 真核生物具有降解含有一种 提前终止密码子。无义介导的RNA衰变过程 (NMRD)在酵母中研究得最好，但在某些方面 NMRD在哺乳动物中似乎有很大的不同。特别是，作为博士。 Dietz发现了(其他人随后也证实了)过早的胡说八道 密码子可以改变核转录因子的剪接模式。一个吸引人的想法是 NMRD演变为一种监视机制，以保护细胞免受有害物质的伤害 截短蛋白的影响；酵母和哺乳动物之间的差异可能 然后反映哺乳动物内含子的丰富和内含子的匮乏 放在酵母里。Dietz博士已经为这一点获得了很好的证据 马凡综合征，其中羧基末端截短纤维蛋白(FBN1) 干扰细胞外基质的组织。这个机制是通过 哪种剪接(可能是核过程)会受到翻译的影响 (可能是细胞质过程)是一个谜，许多模型都是 被引用来探索这一观察。也许关键的问题是关于 NMRD是指核糖体或其他某种装置是否对 扫描ORF；抑制子tRNA部分抑制的能力 NMRD主张发挥核糖体的作用，但奇怪的事情已经发生了。三 被称为UPF1、2和3的酵母蛋白(代表上移码)已经被 基因上被认为是NMRD的必需品；Dietz博士现在克隆了 UPF1的人类同源物，并命名为RENT1(用于无稽之谈的调节 文字记录)。此应用程序旨在探索以下三个方面 RENT1功能：(1)RENT1在小鼠中表达的地点、时间和方式； 以及假定的RENT1的显性负突变(以已知 酵母UPF1p中的显性负值)通过稳定报告结构 无意义突变？(2)小鼠RENT1基因敲除的表型是什么？ NMRD在整个动物体内的缺失是否会影响各种基因的表达 天然的还是人造的mRNA？作为内部控制，以监控 NMRD，Dietz博士将使用一种被称为Gusmps的自发移码突变 这会导致小鼠β-葡萄糖醛酸酶mRNA水平下降200倍。 与R11X无义突变相似的小鼠对照 4-羟基苯丙酮酸双加氧酶基因也被提出。(3)在 组织培养或小鼠模型可通过RENT1下调NMRD的表达 显性负性抢救由过早的废话引起的疾病表型 密码子，甚至被用来检测新的疾病基因？D836X胡说八道 囊性纤维化跨膜中的突变(或类似突变) 电导通道)将提供一个治疗上重要的测试案例 监督这种方法的成功。此外，使用等基因 表达或不表达显性负RENT1等位基因的成纤维细胞系， Dietz博士将尝试通过RDA(表征差异)来重新建立 分析)无意义的密码子是导致燕麦缺陷的原因 回旋性萎缩患者。

DESCRIPTION (adapted from application): All organisms from eubacteria to eukaryotes have mechanisms for degrading transcripts that contain a premature termination codon. The process of nonsense-mediated RNA decay (NMRD) has been best studied in the yeast S. cerevisiae, but some aspects of NMRD appear to be significantly different in mammals. In particular, as Dr. Dietz discovered (and others have subsequently confirmed) premature nonsense codons can alter nuclear mRNA splicing patterns. An attractive idea is that NMRD evolved as a surveillance mechanism to protect cells from the harmful effects of truncated proteins; differences between yeast and mammals might then reflect the abundance of introns in mammals and the paucity of introns in yeast. Dr. Dietz has obtained good evidence for this in the case of Marfan's syndrome, where carboxyterminal truncated fibrillin protein (FBN1) interferes with organization of the extracellular matrix. The mechanism by which splicing (presumably a nuclear process) can be affected by translation (presumably a cytoplasmic process) is a mystery, and many models have been invoked to explore this observation. Perhaps the key question regarding NMRD is whether the ribosome or some other device is responsible for scanning the ORFs; the ability of a suppressor tRNA to partially suppress NMRD argues for a ribosomal role, but stranger things have happened. Three yeast proteins called UPF1, 2, and 3 (for up-frameshift) have been identified genetically as essential for NMRD; Dr. Dietz has now cloned the human homologue of UPF1, and named it RENT1 (for regulation of nonsense transcripts). This application is designed to explore three aspects of RENT1 function: (1) Where, when, and how is RENT1 expressed in the mouse, and will putative dominant negative mutants of RENT1 (modeled on known dominant negatives in yeast UPF1p) stabilize reporter constructs with nonsense mutations? (2) What is the phenotype of a mouse RENT1 knockout, and does loss of NMRD in the whole animal affect expression of various natural or artificial mRNAs? As an internal control to monitor levels of NMRD, Dr. Dietz will use a spontaneous frameshift mutation known as gusmps which causes a 200-fold decrease in murine beta-glucuronidase mRNA levels. Similar controls with the R11X nonsense mutation in the murine 4-hydroxyphenylpyruvic acid dioxygenase gene are also proposed. (3) In a tissue culture or mouse model, can down regulation of NMRD by a RENT1 dominant negative rescue a disease phenotype caused by a premature nonsense codon, or even be used to detect new disease genes? The D836X nonsense mutation (or similar mutations) in the CFTR (cystic fibrosis transmembrane conductance channel) will provide a therapeutically important test case to monitor the success of this approach. In addition, using isogenic fibroblast lines that do or do not express a dominant negative RENT1 allele, Dr. Dietz will attempt to "reestablish" by RDA (representational difference analysis) that a nonsense codon is responsible for the OAT defect in a gyrate atrophy patient.