Characterization of C elegans laf-1: a regulatory ncRNA

线虫 laf-1 的表征：一种调节性 ncRNA

• 批准号: 6869247
• 负责人: ELIZABETH B. GOODWIN
• 金额: $ 31.56万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6869247
• 关键词: Caenorhabditis elegans; RNA splicing; biochemical evolution; developmental genetics; embryogenesis; gene expression; gene induction /repression; genetic regulatory element; genetic screening; helminth genetics; intermolecular interaction; mitotic spindle apparatus; molecular cloning; nucleic acid metabolism; nucleic acid sequence; regulatory gene; reproductive development

DESCRIPTION (provided by applicant): The identification of the large class of miRNAs in plants, vertebrates, and invertebrates, as well as, the characterization of many ncRNAs in bacteria has revolutionized how we think of ncRNAs in biology. Do other classes exist? Identification and characterization of other families of ncRNAs is essential for understanding how general is ncRNA based control. C. elegans hermaphrodites are somatically female; however, the germline produces sperm then oocytes. Hermaphrodite spermatogenesis requires the translational repression of tra-2 by two 3'untranslated elements (called TGEs). laf-1 acts through the TGEs to inhibit tra-2 activity. It is also an essential gene suggesting it regulates other mRNAs besides tra-2. We have cloned laf-1 and find it encodes a not previously described type ncRNA that appears to be produced by the splicing of a precursor Pol lII transcript. We have identified similar ncRNAs in C. briggsae and mouse indicating this class of ncRNAs is broadly conserved. In the next five years, we propose to investigate the expression and processing of this class of ncRNAs. We will use genetics, transgenes and molecular biology to ask what are the cis-acting elements and trans-acting factors required for splicing. Moreover, we will use transgenes and molecular biology to investigate how TRA-1 influences laf-] metabolism. Our data finds that TRA-1 affects the size of the laf-1 ncRNA, suggesting it is required for expression, processing, or stability of laf-1. In addition, we will use RNAi, RNA in situ and molecular analyses to ask how laf-1 interacts with the Wnt and SRC-1 pathways. Preliminary data is consistent with laf-1 acting downstream, upstream or in parallel to the Wnt and/or SRC-1 pathways to control germline sexual development and spindle orientation of the ABar blastomere in the early embryo. We will also screen for suppressors of laf-1 to identify genes required for expression and processing, as well as, potential laf-1 targets. We have already identified a mutation that suppresses the laf-1 lethality. We will clone and characterize this gene. In addition, we will continue to screen for more laf-1 supressors. Using computational and molecular biology, we will look for other members of this ncRNA family. The health relatedness of this work derives from its contribution to an understanding of fundamental control of gene expression. Understanding how this family of ncRNAs is expressed and processed may result in the ability to engineer ncRNAs that could regulate mRNAs of ones choosing, which would have health care applications.

描述（由申请人提供）：鉴定植物，脊椎动物和无脊椎动物中的大型miRNA，以及细菌中许多NCRNA的表征彻底改变了我们对生物学中NCRNA的看法。是否存在其他类别？对其他NCRNA家族的识别和表征对于理解一般基于NCRNA的控制是必不可少的。秀丽隐杆线虫雌雄同体在体外是女性。但是，种系会产生精子，然后产生卵母细胞。雌雄同体的精子发生需要两个3'untranslated元素对Tra-2的翻译抑制（称为TGES）。 LAF-1通过TGE起作用以抑制Tra-2活性。这也是一个重要的基因，表明它调节除了Tra-2之外还具有其他mRNA。我们已经克隆了LAF-1，发现它编码了先前描述的NCRNA类型，该类型似乎是由前体Pol Lii转录本的剪接产生的。我们已经确定了briggsae和小鼠中的类似NCRNA，表明这类NCRNA是广泛保守的。在接下来的五年中，我们建议研究此类NCRNA的表达和处理。我们将使用遗传学，转基因和分子生物学来询问剪接所需的顺式作用元素和跨性因子。而且，我们将使用 转基因和分子生物学研究Tra-1如何影响LAF-]代谢。我们的数据发现TRA-1会影响LAF-1 NCRNA的大小，这表明这是LAF-1的表达，处理或稳定性所必需的。此外，我们将使用RNAi，RNA原位和分子分析来询问LAF-1如何与Wnt和SRC-1途径相互作用。初步数据与LAF-1在下游，上游或与Wnt和/或SRC-1途径平行的作用一致，以控制早期胚胎中Abar Blastomere的种系性发育和主轴取向。我们还将筛选LAF-1的抑制剂，以识别表达和加工所需的基因以及潜在的LAF-1靶标。我们已经确定了一种抑制LAF-1致死性的突变。我们将克隆并表征该基因。此外，我们将继续筛选更多LAF-1级别的supressors。使用计算和分子生物学，我们将寻找该NCRNA家族的其他成员。这项工作的健康相关性来自其对基本控制基因表达的基本控制的贡献。了解如何表达和处理这个NCRNA家族可能会导致能够设计可以调节选择的mRNA的NCRNA，这将具有医疗保健应用。