Elucidating CIS-Acting Elements Regulating Alcohol-Induced Alternative Splicing

描述(申请人提供)：选择性剪接是基因调控的一个重要水平，也是人类和其他哺乳动物蛋白质组多样性的主要来源。至少74%的人类基因通过外显子或外显子片段的选择性剪接表达多个mRNAs。剪接过程的解除调控与各种疾病状态和条件有关，超过60%的已知致病突变会破坏剪接。我们的初步数据表明，酒精处理大鼠肝癌细胞后，许多RNA结合蛋白的表达发生了变化，包括一些已知的剪接活性蛋白。因此，酒精或其新陈代谢很可能会改变剪接。我们将测试这一假设，即不同的顺式作用元件组合决定了酒精诱导的选择性剪接模式。我们将使用外显子阵列来检测乙醇对代谢乙醇的大鼠肝癌细胞和暴露于乙醇的大鼠肝脏中剪接的整体模式的影响。基于这些数据，我们将开发一个计算模型来识别调节这一过程的顺式作用RNA元件。我们将研究乙醇对人肝癌细胞剪接的影响，以确定这些顺式作用的RNA元件是否保守。这项研究的结果不仅将描述酒精如何影响全球剪接模式，而且还将提供一组关于乙醇反应的顺式作用序列的可测试假说。在这个探索性项目中，我们将通过以下三个具体目标来验证我们的假设：(1)确定酒精诱导的大鼠肝癌细胞和大鼠肝脏中替代的前-mRNA剪接；(2)计算发现酒精诱导的顺式作用调节元件；以及(3)测试该模型在人类细胞中的推广。 公共卫生相关性：我们将使用实验和计算方法相结合的方法来研究酒精治疗导致的肝细胞选择性剪接，并揭示酒精诱导的选择性剪接可能导致肝脏损伤的分子机制，特别是顺式作用元件。

DESCRIPTION (provided by applicant): Alternative splicing is an important level of gene regulation and a major source of proteome diversity in humans and other mammals. At least 74% of human genes express multiple mRNAs through alternative splicing of exons or exon segments. Deregulation of the splicing process is associated with a variety of disease states and conditions, and over 60% of known disease- causing mutations disrupt splicing. Our preliminary data indicate that alcohol treatment of rat hepatoma cells causes changes in the expression of many RNA-binding proteins, including some with known splicing activity. Therefore, it is likely that alcohol or its metabolism alters splicing. We will test the hypothesis that distinct combinations of cis-acting elements dictate alcohol-induced patterns of alternative splicing. We will use exon arrays to examine the effects of ethanol on the global pattern of splicing in rat hepatoma cells that metabolize ethanol, and in livers of rats exposed to ethanol. Based upon these data, we will develop a computational model to identify cis-acting RNA elements that regulate this process. We will examine the effects of ethanol on splicing in human hepatoma cells to determine whether these cis-acting RNA elements are conserved. The outcome of this study will not only provide a description of how alcohol affects the global splicing pattern, but also provide a set of testable hypotheses about the cis-acting sequences responsive to ethanol. In this exploratory project, we will test our hypothesis by the following three specific aims: (1) Identify alcohol-induced alternative pre- mRNA splicing in rat hepatoma cells and rat liver; (2) Computational discovery of alcohol- induced cis-acting regulatory elements; and (3) Test the generalization of the model to human cells. PUBLIC HEALTH RELEVANCE: We will use a combination of experimental and computational approaches to investigate alternative splicing in liver cells as a result of alcohol treatment, and to reveal molecular mechanisms, particularly cis-acting elements, that underlie alcohol-induced alternative splicing, which might contribute to liver injury.