Genetic Analysis of Conserved microRNAs in C. elegans

描述(申请人提供)：microRNAs(MiRNAs)是动物发育和生理所需的不可或缺的基因表达调节因子。此外，miRNAs还与多种人类疾病有关，特别是心血管疾病、神经退行性疾病、糖尿病和癌症。然而，只有一小部分单个miRNAs的生物学功能被描述。识别由单个miRNAs直接调控的途径和过程以及识别特定的miRNA靶标对于了解它们在动物发育和人类疾病中的作用至关重要。线虫作为一种在遗传上非常容易驯化的动物，为研究miRNAs的功能提供了一个理想的模型系统，特别是因为许多miRNAs在蠕虫和人类之间显示出完全或接近完全的保守性。在线虫中，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发育正常。MiRNAs很可能与其他miRNAs一起发挥作用，并与其他调控因子一起控制发育途径。为了揭示这种复杂的相互作用，我们研究了在遗传敏化背景下单个miRNAs突变的影响。我们已经在遗传背景中发现了依赖miRNA的表型，由于ArgAerte编码基因alg-1的丢失，总的miRNA活性较低。在这个提案中，我们将分析一个依赖于miRNA的表型，它与五个miRNAs的丢失有关。该建议的目的是确定这五个抑制因子miRNAs调控的生物学途径和直接的mRNA靶点。这项建议的具体目的是：1)确定抑制因子miRNAs发挥作用的遗传途径。为此，我们将进行遗传分析，以测试与发育计时基因、miRNA途径基因和单个miRNA基因的相互作用，这些基因的功能已经被描述，并2)表征依赖miRNA抑制alg-1发育计时缺陷的分子机制。为此，我们将确定抑制子miRNAs是否影响miRNAs的生物发生或活性，检查候选的miRNA靶标，并进行转录组分析，以确定缺少抑制子miRNA活性的蠕虫中调控错误的基因的图谱。与公共卫生相关：这项提案侧重于在蠕虫和哺乳动物之间保守的miRNAs。实现该项目的目标将提供有关在发育过程中受miRNAs调控的通路的关键信息，这些通路可能在人类疾病中被错误调控，并进一步描述miRNA对动物靶标mRNAs调控的生物学原理。 与公共卫生相关：microRNAs是动物发育和生理所需的不可或缺的基因表达调节器。此外，microRNAs还与多种人类疾病有关，特别是心血管疾病、神经退行性疾病、糖尿病和癌症。目前尚不清楚microRNA活性的变化是人类疾病的原因还是结果。确定单个microRNAs的生物学功能对于了解它们在动物发育和人类疾病中的作用至关重要。

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.