Linkage of Nonsense Codons and RNA Splicing

真核细胞的转录后事件是分区化的。转录本在细胞核中剪接，然后转移到细胞质中，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，而且影响核相关事件。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）。通过实验证明RNA剪接的重要性，进一步表明细胞核可能参与PMU。本研究的目的是了解无义密码子如何调节核事件，这些密码子仅被细胞质翻译机制读取。t细胞受体- β (TCRBeta)基因将被用于这项研究，因为它通常在正常的t细胞发育过程中获得ptc，因此监测该基因中ptc获取的机制可能对正常的免疫细胞功能至关重要。在这个项目中，将研究PMU的机制。本研究的目的是：(1)阐明所涉及的特定转录后机制（例如，选择性剪接位点选择与RNA稳定性调节）；(2)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 blot和逆转录酶(RT)-聚合酶链反应（PCR）分析转录的mrna。四环素调控启动子将用于RNA半衰期研究。蛋白质是利用从DNA复制到RNA的信息在细胞的细胞质内合成的。存在细胞监视机制，以确保有缺陷的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的rna（无义密码子）不积累的过程，而另一种形式的mRNA，其中错误已被另一种加工事件消除，积累到高于正常水平。这项调查很重要，原因如下：(1)通过替代加工跳过有害无义密码子的机制可能在生理学上很重要，因为它允许从其他缺陷基因中表达功能蛋白；(2)该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一个组成部分；(3)对无义密码子如何影响核事件的理解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.