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.
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.
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