A role for microRNAs in Drosophila alternative pre-mRNA splicing

描述（由申请人提供）：在后生动物中，选择性前mrna剪接是调节基因表达的一种常见机制。事实上，RNA加工是基因组信息转化为蛋白质组信息的通道。事实上，现在已经认识到30-40%的已知人类和小鼠疾病基因突变影响剪接过程。因此，了解内含子如何被识别以及如何建立选择性剪接模式可能会允许治疗干预。前mRNA中的顺式作用序列元件，被称为沉默子和增强子，已知可调节剪接位点的使用。研究表明，MicroRNAs在翻译和RNA稳定性水平上控制基因表达，一些MicroRNAs（以及piRNAs或rasiRNAs）在细胞核中作为核糖核蛋白（RNP）复合物与arognaute/piwi家族蛋白一起作用，改变染色质结构/组织并控制可移动遗传元件的活性/表达。这项研究的总体目标是询问microRNAs是否可以通过基因表达和剪接连接微阵列来控制果蝇的pre-mRNA剪接模式。我们的工作将集中在使用替代剪接连接微阵列以及microRNA阻断或过表达方法来询问当单个microRNA的功能受到干扰时，全局剪接模式如何变化。为了实现我们的总体目标，我们将：在阻断microRNA功能、阻断microRNA成熟或在细胞培养中使用microRNA过表达后，使用果蝇剪接连接微阵列分析对备选pre-mRNA剪接模式的全基因组影响。2. 在使用dicer-1， dicer-2和ago-2突变体阻断整个动物的microRNA功能或成熟后，使用果蝇剪接连接微阵列分析对替代pre-mRNA剪接模式的全基因组影响。在后生动物中，替代前mrna剪接是调节基因表达的一种常见机制。研究表明，MicroRNAs在翻译和RNA稳定性水平上控制基因表达，一些MicroRNAs（以及piRNAs或rasiRNAs）在细胞核中作为核糖核蛋白（RNP）复合物与arognaute/piwi家族蛋白一起作用，改变染色质结构/组织并控制可移动遗传元件的活性/表达。这项研究的总体目标是询问microRNAs是否可以通过基因表达和剪接连接微阵列来控制果蝇的pre-mRNA剪接模式。

DESCRIPTION (provided by applicant): Alternative pre-mRNA splicing is a common mechanism for regulating gene expression in metazoans. Indeed, RNA processing is a conduit through which genomic information is transferred to proteomic information. In fact, it is now recognized that 30-40% of the known human and mouse disease gene mutations affect the splicing process. Thus, understanding how introns are recognized and how patterns of alternative splicing are set up may allow therapeutic intervention. Cis-acting sequence elements in the pre- mRNA, known as silencers and enhancers, are known to modulate splice site use. MicroRNAs have been shown to control gene expression at the levels of translation and RNA stability and some microRNAs (and piRNAs or rasiRNAs) act in the nucleus as ribonucleoprotein (RNP) complexes with arognaute/piwi family proteins to alter chromatin structure/organization and control the activity/expression of mobile genetic elements. The overall objective of the research proposed in this grant is to ask if microRNAs can control alternative pre-mRNA splicing patterns in Drosophila using gene expression and splice junction microarrays. Our efforts will be focused on the use of alternative splice junction microarrays along with microRNA blocking or over-expression methods to ask how global splicing patterns change when the function of a single microRNA is perturbed. In order to accomplish our overall objectives, we will: 1. Analyze the genome-wide effects on alternative pre-mRNA splicing patterns using Drosophila splice junction microarrays after blocking microRNA function, blocking microRNA maturation or by using microRNA over-expression in cell culture. 2. Analyze the genome-wide effects on alternative pre-mRNA splicing patterns using Drosophila splice junction microarrays after blocking microRNA function or maturation in whole animals using dicer-1, dicer-2 and ago-2 mutants. PUBLIC HEALTH RELEVANCE Alternative pre-mRNA splicing is a common mechanism for regulating gene expression in metazoans. MicroRNAs have been shown to control gene expression at the levels of translation and RNA stability and some microRNAs (and piRNAs or rasiRNAs) act in the nucleus as ribonucleoprotein (RNP) complexes with arognaute/piwi family proteins to alter chromatin structure/organization and control the activity/expression of mobile genetic elements. The overall objective of the research proposed in this grant is to ask if microRNAs can control alternative pre-mRNA splicing patterns in Drosophila using gene expression and splice junction microarrays.