Linkage of Nonsense Codons and RNA Splicing

真核细胞中的9808936个Wilkinson转录后事件被区隔。转录本在细胞核内拼接，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，提前终止密码子(PTCs)不仅影响细胞质事件，还影响与核相关的事件。一些PTC上调已切除PTC的选择性剪接(ALT)mRNAs，这一过程称为PTC介导的上调(PMU)。实验证明了RNA剪接的重要性，进一步表明核可能参与PMU。这项研究的目的是了解只有细胞质翻译机制才知道的无意义密码子是如何调节核事件的。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PTCs，因此监控该基因中PTCs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，将研究PMU的作用机制。本研究的目的是：(1)阐明特定的转录后机制(例如，选择性剪接位点选择与RNA稳定性的调节)和(2)对解释PMU的顺式和反式模型进行实验评估。为了解决这些问题，基因工程TCR构建体将被导入哺乳动物细胞，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)分析转录的mRNAs。四环素(TET)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中的信息在细胞胞浆中合成的。细胞监测机制的存在是为了确保缺陷RNA不会积累，这将导致突变蛋白质的合成。这个项目将研究含有错误(无意义密码子)的RNA不积累的过程，而替代形式的RNA，其中错误已被替代处理事件消除，积累到比正常水平更高的水平。这项研究之所以重要，有几个原因：(1)通过替代加工跳过有害的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缺陷基因的功能蛋白的表达；(2)这一过程可能是识别和销毁含有无义密码子的异常转录本的一般监测机制的组成部分；(3)了解无义密码子如何影响核事件可能会改变人们对基因表达的普遍看法。

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.