REGULATION OF SPLICE SITE CHOICE IN C. ELEGANS

线虫剪接位点选择的调控

• 批准号: 6520290
• 负责人: ALAN M ZAHLER
• 金额: $ 23.3万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6520290
• 关键词: Caenorhabditis elegans; RNA splicing; computer assisted sequence analysis; developmental genetics; genetic regulation; genetic regulatory element; green fluorescent proteins; nucleic acid sequence; precursor mRNA; reporter genes; transcription factor

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.

前体信使RNA剪接是基因表达的一个重要步骤，在高等生物中可以通过选择性剪接进行高度调节。 虽然生物化学方法已成功地确定蛋白质因子参与调控的剪接位点选择的后生动物，总是有关注，在体外方法，使用细胞系提取物不确定真正的发育和组织特异性调节剪接位点的选择。 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，这可能会揭示特定的调节因子。 C.秀丽线虫为我们提供了一个令人兴奋的研究剪接及其调节的机会。 完整的基因组序列和超过100，000个cDNA序列为计算机方法识别和表征剪接调控序列提供了独特的机会。 发达的遗传系统可用于鉴定调控特异性剪接的基因。 分子转化和细胞学工具，如使用绿色荧光蛋白，提供了机会，工程报告基因，通过剪接可以监测与视觉和分子手段。 在这个计划中，我们提出了一个遗传，分子和生物信息学的方法来分析剪接调控在C。优美的 具体目标是：1-鉴定和表征参与隐蔽剪接位点选择的调节的基因。 这些类型的基因可以在人类疾病显示中产生重要的后果。 2-使用一种新的遗传筛选，包括通过使用绿色荧光蛋白报告子观察可变剪接，以鉴定参与let-2基因可变剪接发育调控的反式作用因子和顺式调控元件。 3-使用我们在C中通过计算确定的845个可变剪接基因的集合。elegans来计算识别前mRNA中可能参与剪接调控的顺式元件。 这些推定的元素将使用我们已经开发的体内剪接报告方法进行表征。