Linkage of Nonsense Codons and RNA Splicing

9808936威尔金森 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，然后易位到细胞质，在那里发生翻译。 一个令人惊讶的观察结果是，提前终止密码子（PTC）不仅影响细胞质事件，但也核相关的事件。 一些PTC上调已经切除PTC的选择性剪接（alt）mRNA，这一过程称为PTC介导的上调（PMU）。 细胞核可能参与PMU，这一点在证明RNA剪接重要性的实验中得到了进一步的证实。 本研究的目的是了解无义密码子，这是唯一已知的细胞质翻译机器阅读，调节核事件。 T细胞受体β（TCRBeta）基因将用于本研究，因为它通常在正常T细胞发育期间获得PTC，因此监测该基因中PTC获得的机制可能对正常免疫细胞功能至关重要。 在本项目中，负责PMU的机制将进行研究。 本研究的目的是：（1）阐明所涉及的特定转录后机制（例如，选择性剪接位点与调节RNA稳定性）和（2）实验评估解释PMU的顺式和反式模型。 为了解决这些问题，将基因工程改造的TCR构建体转染到哺乳动物细胞中，并通过核糖核酸酶（Rnase）保护、北方印迹和逆转录酶（RT）-聚合酶链式反应（PCR）分析来分析转录的mRNA。 四环素（泰特）调节的启动子将用于RNA半衰期研究。 蛋白质是利用从DNA复制到RNA的信息在细胞的胞质溶胶中合成的。 存在细胞监视机制以确保将导致突变蛋白质合成的缺陷RNA不积累。 该项目将研究含有错误（无义密码子）的RNA不积累的过程，而替代形式的mRNA，其中错误已被替代加工事件消除，积累到高于正常水平。 这项调查很重要，原因如下：（1）负责通过替代加工跳过有害的无义密码子的机制可能是生理学上重要的，因为它可以允许功能蛋白质从其他缺陷基因表达，（2）该过程可能是识别和破坏含有无义密码子的异常转录物的一般监视机制的组成部分，以及（3）理解无义密码子如何影响核事件可能会改变基因表达的主流观点。

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.