A role for microRNAs in Drosophila alternative pre-mRNA splicing

描述(由申请人提供)：替代的前-信使核糖核酸剪接是调节后生动物基因表达的常见机制。的确，RNA处理是基因组信息传递到蛋白质组信息的管道。事实上，现在已经认识到，已知的人类和小鼠疾病基因突变中有30%-40%会影响剪接过程。因此，了解内含子是如何识别的，以及选择性剪接模式是如何建立的，可能会允许进行治疗干预。已知Pre-mRNA中的顺式作用序列元件称为沉默和增强子，调节剪接位点的使用。已有研究表明，microRNAs在翻译和RNA稳定性水平上控制基因的表达，一些microRNAs(和piRNAs或rasiRNAs)在细胞核内以核糖核蛋白(RNP)的形式与arognaute/piwi家族蛋白作用，改变染色质的结构/组织，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中不同的前mRNA剪接模式。我们的努力将集中在使用替代剪接连接微阵列以及microRNA阻断或过度表达的方法来研究当单个microRNA的功能受到干扰时，全球剪接模式是如何变化的。为了实现我们的总体目标，我们将：1.利用果蝇剪接连接微阵列，分析阻断microRNA功能、阻断microRNA成熟或在细胞培养中过度表达microRNA后，全基因组范围内对不同的前mRNA剪接模式的影响。2.利用DICER-1、DICER-2和AGO-2突变体，利用果蝇剪接连接微阵列，分析了在整个动物体内阻断microRNA功能或成熟后，全基因组对不同的前-mRNA剪接模式的影响。公共卫生相关性替代前-mRNA剪接是调节后生动物基因表达的一种常见机制。已有研究表明，microRNAs在翻译和RNA稳定性水平上控制基因的表达，一些microRNAs(和piRNAs或rasiRNAs)在细胞核内以核糖核蛋白(RNP)的形式与arognaute/piwi家族蛋白作用，改变染色质的结构/组织，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中不同的前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.