3 UTR CONTROL OF TRA-2 TRANSLATION IN C ELEGANS

3 线虫中 TRA-2 翻译的 UTR 控制

• 批准号: 6342900
• 负责人: ELIZABETH B. GOODWIN
• 金额: $ 26.63万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6342900
• 关键词: Caenorhabditis elegans; RNA; developmental genetics; gel mobility shift assay; gene expression; gene mutation; genetic regulation; genetic regulatory element; genetic translation; messenger RNA; molecular cloning; nucleotides; reporter genes; sex determination

DESCRIPTION:This is the first renewal application from a beginning investigator, Dr. Elizabeth Goodwin of Northwestern University. Dr. Goodwin is seeking five years of support to continue her studies of the translational regulation of the tra-2 mRNA in the nematode C. elegans. tra-2 is a member of a cascade of genes that controls C. elegans' sexual identity. The presence of the tra-2 gene product, a large transmembrane protein, promotes female cell fates (in XX animals). Translation and poly(A) length of the tra-2 mRNA are regulated by two 28 nt elements, DREs, located in the mRNA's 3'-UTR. These elements are specifically bound by a factor designated DRF which is thought to repress translation and limit poly(A) length. Three genes, laf-1, tra-3, and tra-1 regulate DRE control of tra-2 expression. laf-1 is required for translational repression, and has thus been a candidate for DRF. tra-3 and tra-1 act to free tra-2 from DRE regulation. Interestingly, tra-1 also appears to be a transcriptional regulator in the sex determination pathway. Yet another gene, glp-1, regulates tra-2 translation by a DRE-independent pathway. The proposed experiments will attempt to elucidate the mechanism by which tra-2 translation is governed by these factors and its 3'-UTR. Seven specific aims will address three general questions: 1) What are the precise cis-acting elements and how do they regulate translation? This question will be addressed by using reporter assays, mutational analysis, and RNA gel shift assays to identify the nucleotides required for DRE and glp-1 regulation, to determine whether glp-1 regulation also affects poly(A) length, and to test whether tra-1 expression is also regulated by 3' DREs or other UTR elements. 2) What gene products regulate translation and how? Here, the PI will utilize molecular (e.g., three-hybrid) and biochemical techniques to identify, clone, and characterize DRF and laf-1. Subsequently, genetics, reporter constructs, and gel shifts will be employed to characterize the mechanisms by which tra-1, tra-3, and glp-1 regulate tra-2 translation and to identify the genes that affect tra-1 translational control. 3) How do 3'-UTR controls regulate translation in a manner that allows correct temporal and spatial regulation of developmental fate? Here, the PI will use genetics, reporter constructs, and gel shift analyses to determine how laf-1, tra-3, tra-1, and glp-1 control the pattern of tra-2 expression during development.

描述：这是从一开始的第一个续订应用程序 调查员，西北大学的伊丽莎白·古德温博士。 古德温博士 正在寻求五年的支持，以继续她的研究 线虫秀丽隐杆线虫中Tra-2 mRNA的翻译调节。 Tra-2是控制秀丽隐杆线虫的基因级联的成员 身份。 Tra-2基因产物的存在，一个大型跨膜 蛋白质，促进雌性细胞命运（在XX动物中）。 翻译和 poly（a）Tra-2 mRNA的长度由两个28 nt元素DRE，DRE， 位于mRNA的3'-UTR中。 这些元素是由 被认为是压制翻译和限制的因子指定的DRF poly（a）长度。 LAF-1，TRA-3和TRA-1的三个基因调节DRE控制 Tra-2表达。 LAF-1是翻译抑制所必需的，并且 因此，曾是DRF的候选人。 tra-3和tra-1行为使tra-2从 DRE调节。 有趣的是，Tra-1似乎也是转录 性别确定途径中的调节器。 另一个基因GLP-1， 通过独立于DRE的途径调节Tra-2翻译。 拟议的实验将尝试阐明 Tra-2翻译受这些因素及其3'-UTR的控制。 七 具体目的将解决三个一般问题：1）确切的问题是什么 顺式作用元素，它们如何调节翻译？ 这个问题 将通过使用记者测定，突变分析和RNA凝胶来解决 换档测定以识别DRE和GLP-1所需的核苷酸 调节，以确定GLP-1调节是否也影响poly（a） 长度，并测试Tra-1表达是否也受3'DRE或 其他UTR元素。 2）哪些基因产品调节翻译？如何调节？ 在这里，PI将利用分子（例如三杂化）和生化 识别，克隆和表征DRF和LAF-1的技术。 随后，将采用遗传学，记者结构和凝胶转移 为了表征Tra-1，Tra-3和GLP-1调节的机制 Tra-2翻译并识别影响Tra-1翻译的基因 控制。 3）3'-UTR如何以一种方式控制翻译 允许正确的时间和空间调节发展命运？ 这里， PI将使用遗传学，报告基因构建体和凝胶转移分析到 确定LAF-1，TRA-3，TRA-1和GLP-1如何控制Tra-2的模式 发育过程中的表达。