NONSENSE RNA SURVEILLANCE IN HEALTH AND DISEASE

描述（根据应用改编）：从真细菌到 真核生物有降解转录本的机制， 提前终止密码子 无义介导的RNA衰变过程 （NMRD）在酵母S.酿酒，但某些方面的 NMRD似乎在哺乳动物中显著不同。 特别是，作为博士。 迪茨发现了（其他人随后证实）不成熟的废话 密码子可以改变核mRNA剪接模式。 一个吸引人的想法是， NMRD作为一种监视机制发展，以保护细胞免受有害物质的侵害。 截短蛋白质的影响;酵母和哺乳动物之间的差异可能 反映了哺乳动物中内含子的丰富和内含子的缺乏， 在酵母中。 Dietz博士已经获得了很好的证据， 马凡氏综合征，其中羧基末端截短的β-胡萝卜素蛋白（FBN 1） 干扰细胞外基质的组织。 的机制 哪种剪接（可能是核过程）会受到翻译的影响 （可能是一个细胞质过程）是一个谜，许多模型已经被证实。 来探讨这一观察。 也许关键问题在于 NMRD是核糖体或其他装置负责 扫描ORF;抑制tRNA部分抑制 NMRD主张核糖体的作用，但更奇怪的事情发生了。 三 被称为UPF 1、2和3的酵母蛋白（用于上移码）已经被 在基因上被确定为对匪RD至关重要; Dietz博士现在已经克隆了 UPF 1的人类同源物，并将其命名为RENT 1（用于调节无义 成绩单）。 此应用程序旨在探索以下三个方面： RENT 1功能：（1）RENT 1在小鼠中的何处、何时以及如何表达， 并将RENT 1的推定显性负突变体（以已知的 酵母UPF 1 p中的显性阴性）稳定报告基因构建体， 无意义突变 (2)什么是小鼠RENT 1基因敲除的表型， 以及在整个动物中丧失匪RD是否会影响各种 天然或人工mRNA？ 作为一种内部控制， 全国改良研究部，迪茨博士将使用一种自发的移码突变，称为gusmps 其导致鼠β-葡萄糖醛酸酶mRNA水平降低200倍。 在小鼠中具有R11 X无义突变的类似对照 4-还提出了羟苯基烟酸双加氧酶基因。 (3)中 组织培养或小鼠模型，可以通过RENT 1下调匪RD 显性阴性挽救由过早无义引起的疾病表型 密码子，甚至用于检测新的疾病基因？ D836 X无意义 CFTR（囊性纤维化跨膜）中的突变（或类似突变） 传导通道）将提供治疗上重要的测试案例， 监测这种方法的成功。 此外，使用同基因 表达或不表达显性负性RENT 1等位基因的成纤维细胞系， 博士Dietz将尝试通过RDA（代表性差异）“重建” 无义密码子是导致OAT缺陷的原因。 回旋状萎缩病人。

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.