Genetic Analysis of Conserved microRNAs in C. elegans

描述（由申请人提供）：MicroRNAs （miRNAs）是动物发育和生理所需的不可缺少的基因表达调节剂。此外，mirna与广泛的人类疾病有关，特别是心血管疾病、神经退行性疾病、糖尿病和癌症。然而，只有少数单个mirna的生物学功能被描述。鉴定由单个miRNA直接调控的途径和过程以及鉴定特定的miRNA靶点对于了解它们在动物发育和人类疾病中的作用至关重要。秀丽隐杆线虫作为一种遗传易感的动物，为研究mirna的功能提供了理想的模型系统，特别是许多mirna在蠕虫和人类之间表现出完全或接近完全的保守性。在秀丽隐杆线虫中，大多数mirna不是单独发育所必需的；携带miRNA基因突变的蠕虫基本上正常发育。mirna可能与其他mirna和其他调节因子一起起作用，以控制发育途径。为了揭示这种复杂的相互作用，我们研究了基因敏感背景下单个mirna突变的影响。由于argonaute编码基因alg-1的缺失，我们在遗传背景中发现了miRNA依赖表型，其总体miRNA活性较低。在本提案中，我们将分析与5种mirna缺失相关的单一mirna依赖表型。本提案的目的是确定这五种抑制mirna调控的生物学途径和直接mRNA靶点。本提案的具体目的是：1)确定抑制mirna发挥作用的遗传途径。为此，我们将进行遗传分析，以测试与发育定时基因、miRNA通路基因和单个miRNA基因的相互作用，其中一种功能已经被描述，2)表征miRNA依赖性抑制alg-1发育定时缺陷的分子机制。为了做到这一点，我们将确定抑制miRNA是否影响miRNA的生物发生或活性，检查候选miRNA靶点，并进行转录组分析，以确定缺失抑制miRNA活性的蠕虫中失调基因的特征。公共卫生相关性：本提案侧重于蠕虫和哺乳动物之间保守的mirna。实现该项目的目标将提供miRNA在发育过程中调控的在人类疾病中可能被错误调控的通路的关键信息，并进一步描述miRNA调控动物靶mrna的生物学原理。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are indispensable regulators of gene expression that are required for animal development and physiology. In addition, miRNAs have been implicated in a wide spectrum of human diseases, notably cardiovascular disease, neurodegenerative disease, diabetes, and cancer. However, the biological functions of only a small number of individual miRNAs have been described. The identification of pathways and processes directly regulated by individual miRNAs and the identification of specific miRNA targets is vital to understand their role in animal development as well as in human disease. As an eminently genetically tractable animal, C. elegans provides an ideal model system in which to study the functions of miRNAs, particularly as many miRNAs show complete or near-complete conservation between worms and humans. In C. elegans, most miRNAs are not individually required for development; worms carrying mutations in miRNA genes develop essentially normally. It is likely that miRNAs function with other miRNAs and with additional regulatory factors to control developmental pathways. To reveal such complex interactions, we have examined the effects of mutations in individual miRNAs in genetically sensitized backgrounds. We have identified miRNA- dependent phenotypes in a genetic background with lower overall miRNA activity due to loss of an Argonaute-encoding gene, alg-1. In this proposal, we will analyze a single miRNA-dependent phenotype that is associated with the loss of five miRNAs. The objective of this proposal is to identify the biological pathways and direct mRNA targets regulated by these five suppressor miRNAs. The specific aims in this proposal are to: 1) define the genetic pathway in which suppressor miRNAs function. To do this, we will perform genetic analysis to test for interactions with developmental timing genes, miRNA pathway genes, and individual miRNA genes, for which a function has been described and 2) characterize the molecular mechanism for miRNA-dependent suppression of alg-1 developmental timing defects. To do this, we will determine if suppressor miRNAs affect the biogenesis or activity of miRNAs, examine candidate miRNA targets, and perform transcriptome analysis to identify the profile of misregulated genes in worms missing suppressor miRNA activity. PUBLIC HEALTH RELEVANCE: This proposal focuses on miRNAs conserved between worms and mammals. Achieving the goals of this project will provide key information about pathways regulated by miRNAs during development that may be misregulated in human disease and to further describe the biological principles of miRNA regulation of target mRNAs in animals. PUBLIC HEALTH RELEVANCE: MicroRNAs are indispensable regulators of gene expression that are required for animal development and physiology. In addition, microRNAs have been implicated in a wide spectrum of human diseases, notably cardiovascular disease, neurodegenerative disease, diabetes, and cancer. It is not clear whether changes in microRNA activity are a cause or a consequence of human disease. The identification of biological functions of individual microRNAs is vital to understand their role in animal development as well as in human disease.