REGULATION OF SPLICE SITE CHOICE IN C. ELEGANS

前体信使RNA剪接是基因表达的重要步骤，在高等生物中可以通过选择性剪接高度调控。虽然生化方法已经成功地识别了参与调控后生动物剪接位点选择的蛋白质因子，但人们一直担心，使用细胞系提取物的体外方法并不能识别剪接位点选择的真正发育和组织特异性调节因子。在后生动物模型系统中研究选择性剪接的定向遗传学方法很少，这可能揭示特定的调控因子。线虫为我们提供了一个研究剪接及其调控的令人兴奋的机会。完整的基因组序列和超过100,000个cdna序列为识别和表征剪接调控序列的计算方法提供了一个独特的机会。这个发达的遗传系统可以用来识别调节特定剪接的基因。分子转化和细胞学工具，如绿色荧光蛋白的使用，提供了设计报告基因的机会，通过这些报告基因，可以用视觉和分子手段来监测剪接。在这项建议中，我们提出了一种遗传、分子和生物信息学的方法来分析线虫的剪接调控。其具体目的是：1-识别和鉴定参与隐蔽剪接位点选择调控的基因。这些类型的基因可能会对人类疾病的表现产生重要影响。2-使用一种新的遗传屏幕，包括通过使用绿色荧光蛋白报告对选择性剪接进行可视化，以确定参与let-2基因选择性剪接的发育调控的反式作用因子和顺式调控元件。3-使用我们在线虫中通过计算确定的845个选择性剪接基因的集合，通过计算确定可能参与剪接调控的前mRNA中的顺式元件。这些假定的元件将使用我们开发的体内剪接报告方法进行表征。

Precursor messenger RNA splicing is an essential step in gene expression that can be highly regulated by alternative splicing in higher organisms. While biochemical approaches have been successful in identifying protein factors involved in the regulation of splice site choice in metazoans, there is always the concern that in vitro approaches using extracts from cell lines do not identify the true developmental and tissue-specific regulators of splice site choice. Directed genetic approaches to studying alternative splicing in metazoan model systems, which could reveal the specific regulators, have been few. C. elegans presents us with an exciting opportunity to study splicing and its regulation. The complete genome sequence and over 100,000 cDNA sequences present a unique opportunity for a computational approach to identify and characterize splicing regulatory sequences. The well-developed genetic system could be used to identify genes that regulate specific splicing. The molecular transformation and cytological tools such as the use of green fluorescent protein provide opportunities to engineer reporter genes through which splicing can be monitored with both visual and molecular means. In this proposal we present a genetic, molecular and bioinformatics approach to analyze splicing regulation in C. elegans. The specific aims are to: 1- identify and characterize genes involved in the regulation of cryptic splice site choice. These types of genes can have important consequences in human disease display. 2- use a novel genetic screen, involving visualization of alternative splicing through the use of a green fluorescent protein reporter, to identify trans-acting factors and cis regulatory elements involved in the developmental regulation of alternative splicing of the let-2 gene. 3- use a collection of 845 alternatively spliced genes we have identified computationally in C. elegans to computationally identify cis elements in the pre-mRNA potentially involved in splicing regulation. These putative elements will be characterized using an in vivo splicing reporter method that we have developed.