REGULATION OF SPLICE SITE CHOICE IN C. ELEGANS

线虫剪接位点选择的调控

• 批准号: 6603066
• 负责人: ALAN M ZAHLER
• 金额: $ 23.27万
• 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 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6603066
• 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剪接是基因表达的重要步骤，可以通过较高的生物体中的替代剪接来高度调节。 尽管生化方法已经成功地鉴定出与后生动物中剪接部位选择有关的蛋白质因子，但始终担心使用细胞系提取物的体外方法并不能识别剪接部位选择的真正发育和组织特异性调节剂。 在可能揭示特定调节剂的后生模型系统中研究替代剪接的定向遗传方法很少。 秀丽隐杆线虫为我们提供了一个令人兴奋的学习剪接及其调节的机会。 完整的基因组序列和100,000多个cDNA序列为识别和表征剪接调节序列的计算方法提供了独特的机会。 发达的遗传系统可用于识别调节特定剪接的基因。 分子转化和细胞学工具（例如使用绿色荧光蛋白）为工程师报告基因提供了机会，可以通过视觉和分子均值对剪接进行监测。 在此提案中，我们提出了一种遗传，分子和生物信息学方法，用于分析秀丽隐杆线虫中的剪接调节。 具体目的是：1-识别和表征与密码剪接位点选择调节有关的基因。 这些类型的基因可能在人类疾病显示中产生重要的后果。 2-使用新型的遗传筛选，涉及通过使用绿色荧光蛋白报道器可视化替代剪接的替代剪接，以鉴定跨作用因子和CIS调节元素涉及的替代剪接的LET-2基因的发育调节。 3-使用我们在秀丽隐杆线虫中鉴定的845个剪接基因的集合来识别潜在参与剪接调控的MRNA中的CIS元素。 这些推定的元素将使用我们开发的体内剪接报告基因方法来表征。