Elucidating CIS-Acting Elements Regulating Alcohol-Induced Alternative Splicing

描述（由申请人提供）：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mRNA。剪接过程的失调与多种疾病状态和病症相关，并且超过60%的已知致病突变破坏剪接。我们的初步数据表明，酒精处理大鼠肝癌细胞导致许多RNA结合蛋白表达的变化，包括一些已知的剪接活性。因此，酒精或其代谢可能会改变剪接。我们将检验这一假设，即顺式作用元件的不同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研究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全局模式的影响。基于这些数据，我们将开发一个计算模型，以确定调节这一过程的顺式作用RNA元件。我们将研究乙醇对人类肝癌细胞剪接的影响，以确定这些顺式作用RNA元件是否保守。这项研究的结果不仅将提供一个描述酒精如何影响全球剪接模式，但也提供了一套可检验的假设顺式作用序列响应乙醇。在这个探索性项目中，我们将通过以下三个具体目标来测试我们的假设：（1）鉴定大鼠肝癌细胞和大鼠肝脏中酒精诱导的替代性前mRNA剪接;（2）计算发现酒精诱导的顺式作用调节元件;和（3）测试模型对人类细胞的推广。 公共卫生相关性：我们将使用实验和计算的方法相结合，以调查作为酒精治疗的结果在肝细胞中的选择性剪接，并揭示分子机制，特别是顺式作用元件，酒精诱导的选择性剪接，这可能有助于肝损伤的基础。

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.