Linkage of Nonsense Codons and RNA Splicing

无义密码子与 RNA 剪接的联系

• 批准号: 9808936
• 负责人: Miles Wilkinson
• 金额: $ 28.49万
• 依托单位: University of Texas, M.D. Anderson Cancer Center
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9808936&HistoricalAwards=false
• 关键词: Linkage; Nonsense; Codons; RNA; Splicing

9808936 Wilkinson Post-transcriptional events in eukaryotic cells are compartmentalized. Transcripts are spliced in the nucleus, and then translocated to the cytoplasm, where translation occurs. A surprising observation has been that premature termination codons (PTCs) affect not only cytoplasmic events but also nuclear-associated events. Some PTCs upregulate alternatively spliced (alt) mRNAs that have excised the PTC, a process called PTC-mediated upregulation (PMU). That the nucleus may be involved in PMU is further suggested by experiments demonstrating the importance of RNA splicing. The goal of this study is to understand how nonsense codons, which are only known to be read by the cytoplasmic translational machinery, regulate nuclear events. The T-cell receptor-beta (TCRBeta) gene will be used for this investigation because it commonly acquires PTCs during normal T-cell development and therefore mechanisms that monitor the acquisition of PTCs in this gene may be critical for normal immune cell function. In this project, the mechanism responsible for PMU will be studied. The objectives of the study are: (1) to elucidate the specific post-transcriptional mechanism involved (e.g., alternative splice-site selection vs. regulation of RNA stability) and (2) to experimentally evaluate cis and trans models that explain PMU. To address these issues, genetically engineered TCR constructs will be transfected into mammalian cells and the transcribed mRNAs analyzed by ribonuclease (Rnase) protection, northern blot, and reverse transcriptase (RT)-polymerase chain reaction (PCR) analyses. A tetracycline (tet)-regulated promoter will be used for RNA half-life studies. Proteins are synthesized within the cytosol of the cell using information copied from DNA into RNA. Cellular surveillance mechanisms exist to insure that defective RNAs, which would result in the synthesis of mutant proteins, do not accumulate. This project will investigate the process by which RNAs containing mistakes ( nonsense codons) do not accumulate, while alternative forms of the mRNA, in which the mistake has been eliminated by an alternative processing event, accumulate to a higher than normal level. This investigation is important for several reasons: (1) the mechanism responsible for skipping deleterious nonsense codons by alternative processing may be physiologically important because it can permit the expression of functional proteins from otherwise defective genes, (2) this process may be a component of a general-surveillance mechanism that recognizes and destroys aberrant transcripts containing nonsense codons, and (3) an understanding of how nonsense codons affect nuclear events may alter the prevailing view of gene expression.

9808936 Wilkinson 真核细胞中的转录后事件是区室化的。 转录本在细胞核中剪接，然后转移到细胞质，在那里发生翻译。 一个令人惊讶的观察结果是，过早终止密码子 (PTC) 不仅影响细胞质事件，还影响核相关事件。 一些 PTC 上调切除 PTC 的选择性剪接 (alt) mRNA，这一过程称为 PTC 介导的上调 (PMU)。 证明 RNA 剪接重要性的实验进一步表明细胞核可能参与 PMU。 这项研究的目的是了解仅由细胞质翻译机制读取的无义密码子如何调节核事件。 T 细胞受体-β (TCRbeta) 基因将用于这项研究，因为它通常在正常 T 细胞发育过程中获得 PTC，因此监测该基因中 PTC 获得的机制可能对于正常免疫细胞功能至关重要。 在这个项目中，将研究负责PMU的机制。 该研究的目的是：(1) 阐明所涉及的具体转录后机制（例如，选择性剪接位点选择与 RNA 稳定性调节）；(2) 通过实验评估解释 PMU 的顺式和反式模型。 为了解决这些问题，基因工程 TCR 构建体将被转染到哺乳动物细胞中，并通过核糖核酸酶 (Rnase) 保护、Northern 印迹和逆转录酶 (RT)-聚合酶链式反应 (PCR) 分析来分析转录的 mRNA。 四环素 (tet) 调节的启动子将用于 RNA 半衰期研究。 使用从 DNA 复制到 RNA 的信息在细胞的胞浆内合成蛋白质。 细胞监视机制的存在是为了确保有缺陷的 RNA 不会积累，从而导致突变蛋白的合成。 该项目将研究含有错误（无义密码子）的 RNA 不会累积的过程，而 mRNA 的替代形式（错误已通过替代加工事件消除）会累积到高于正常水平。 这项研究很重要，原因有几个：（1）通过替代处理跳过有害无义密码子的机制可能在生理上很重要，因为它可以允许来自其他有缺陷的基因表达功能性蛋白质，（2）这个过程可能是识别和破坏含有无义密码子的异常转录本的一般监视机制的组成部分，（3）了解无义密码子如何影响核事件可能会改变 基因表达的普遍观点。