Genetic Analysis of Conserved microRNAs in C. elegans

描述（由申请人提供）：MicroRNA（miRNA）是动物发育和生理学所需的基因表达不可或缺的调节因子。此外，miRNA 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，只有少数单个 miRNA 的生物学功能被描述。识别单个 miRNA 直接调控的途径和过程以及特定 miRNA 靶标的识别对于了解它们在动物发育以及人类疾病中的作用至关重要。作为一种在遗传上非常容易驯化的动物，线虫为研究 miRNA 的功能提供了一个理想的模型系统，特别是因为许多 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 并不是发育所必需的；携带 miRNA 基因突变的线虫发育基本正常。 miRNA 很可能与其他 miRNA 以及其他调控因子一起发挥作用来控制发育途径。为了揭示这种复杂的相互作用，我们研究了遗传敏感背景下单个 miRNA 突变的影响。我们已经在遗传背景中鉴定出 miRNA 依赖性表型，由于 Argonaute 编码基因 alg-1 的丢失，总体 miRNA 活性较低。在本提案中，我们将分析与 5 个 miRNA 丢失相关的单一 miRNA 依赖性表型。该提案的目的是确定这五种抑制 miRNA 调节的生物途径和直接 mRNA 靶标。本提案的具体目标是：1) 定义抑制 miRNA 发挥功能的遗传途径。为此，我们将进行遗传分析，以测试与发育时序基因、miRNA 途径基因和单个 miRNA 基因的相互作用，其功能已被描述，并且 2) 表征 miRNA 依赖性抑制 alg-1 发育时序缺陷的分子机制。为此，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 靶标，并进行转录组分析，以确定缺少抑制 miRNA 活性的线虫中失调基因的概况。公共健康相关性：该提案重点关注蠕虫和哺乳动物之间保守的 miRNA。实现该项目的目标将提供有关发育过程中 miRNA 调节的途径的关键信息，这些途径可能在人类疾病中被错误调节，并进一步描述动物中 miRNA 调节靶 mRNA 的生物学原理。 公共健康相关性：MicroRNA 是动物发育和生理学所需的基因表达不可或缺的调节因子。此外，microRNA 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。识别单个 microRNA 的生物学功能对于了解它们在动物发育以及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.