REGULATION OF SPLICE SITE CHOICE IN C. ELEGANS

线虫剪接位点选择的调控

基本信息

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
项目类别：
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

项目摘要

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 剪接是基因表达中的一个重要步骤，可以通过高等生物中的选择性剪接进行高度调控。虽然生化方法已成功地鉴定了参与后生动物剪接位点选择调节的蛋白质因子，但始终存在这样的担忧：使用细胞系提取物的体外方法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。研究后生动物模型系统中选择性剪接的定向遗传方法很少，这种方法可以揭示特定的调节因子。线虫为我们提供了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）提供了设计报告基因的机会，通过该报告基因可以用视觉和分子手段监测剪接。在本提案中，我们提出了一种遗传、分子和生物信息学方法来分析线虫的剪接调控。具体目标是： 1-鉴定和表征参与隐秘剪接位点选择调节的基因。这些类型的基因可能对人类疾病的表现产生重要影响。 2-使用新颖的遗传筛选，包括通过使用绿色荧光蛋白报告基因实现选择性剪接的可视化，以鉴定参与let-2基因选择性剪接发育调节的反式作用因子和顺式调节元件。 3-使用我们在秀丽隐杆线虫中通过计算鉴定的845个选择性剪接基因的集合，通过计算鉴定前mRNA中可能参与剪接调节的顺式元件。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。