Regulation of biogenesis and function of let-7 microRNA in C. elegans

描述（由申请人提供）：MicroRNAs （miRNAs）包括新发现的一类非编码RNA基因，特定的miRNAs已经与与人类健康和疾病相关的关键基因调控作用相关联。let-7基因是这一新型调控分子家族的创始成员，它作为理解控制miRNA生物发生和功能的分子机制的模型。此外，let-7在物种间的保守性非常好，它在人类中作为肿瘤抑制因子的潜在作用与其促进细胞分化的功能有关，这最初是在秀丽隐杆线虫中确定的。本文所述的研究建立在我们对调节let-7 miRNA生物发生的动态过程的识别和我们开发的强大的生化方法的基础上，旨在揭示miRNA复合物（包括argonaute蛋白ALG-1）如何识别和调节体内的直接靶基因。我们的研究对于理解mirna如何跨物种表达和功能以及这些途径的扰动如何导致人类疾病具有广泛的意义。为了验证特定序列和蛋白质协同调节let-7和相关mirna的表达和功能的假设，我们提出以下目标。1. 表征控制let-7家族miRNAs表达的转录后机制。(A)调节let-7 miRNA加工的顺式作用元件将通过体外结构分析和体内表达和功能分析来确定。(B)将定义介导let-7相关mirna （mir-84、mir-48、mir-241和mir-795）转录后调控的主要转录本和元件。2. 确定LIN-28和LIN-42蛋白在调节mirna表达中的作用。(A) lin-28在let-7 miRNA生物发生中的作用将通过体内和体外RNA结合试验进行研究。一个基于RNAi的敏感筛选将被用于识别依赖于let-7活性的lin-28突变表型的增强子和抑制子。(B)周期同源物LIN-42在调节miRNA成熟中的新功能将通过确定RNA靶点和遗传相互作用物来研究。3. 鉴定体内与ALG-1结合的miRNA靶位点及其相互作用的调控结果。(A) ALG-1结合和调节的序列依赖于特定的mirna，将使用一种新的生化和计算方法结合微阵列分析来鉴定。(B)特异性miRNA靶点将通过高通量遗传方法和敏感的报告基因检测来验证。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) comprise a newly discovered class of non-coding RNA genes and specific miRNAs have already been linked to key gene regulatory roles relevant to human health and disease. The let-7 gene is a founding member of this novel family of regulatory molecules and it serves as a model for understanding the molecular mechanisms that control miRNA biogenesis and function. Furthermore, let-7 is exceptionally well conserved across species and its potential role as a tumor suppressor in humans is related to its function in promoting cellular differentiation, originally determined in C. elegans. The research described in this renewal builds upon our identification of dynamic processes that regulate biogenesis of let-7 miRNA and our development of a robust biochemical approach aimed at revealing how the miRNA complex, which includes the argonaute protein ALG-1, recognizes and regulates direct target genes in vivo. Our studies are broadly relevant for understanding how miRNAs are expressed and function across species and also how perturbation of these pathways contributes to human disease. To test the hypothesis that specific sequences and proteins cooperate to regulate expression and function of let-7 and related miRNAs, we propose the following aims. 1. Characterize the post-transcriptional mechanisms that control expression of let-7 family miRNAs. (A) The cis-acting elements that regulate processing of let-7 miRNA will be determined using in vitro structure analyses and in vivo expression and function assays. (B) The primary transcripts and elements that mediate post-transcriptional regulation of let-7 related miRNAs (mir-84, mir-48, mir-241 and mir-795) will be defined. 2. Determine the function of the LIN-28 and LIN-42 proteins in regulating the expression of miRNAs. (A) The role of lin-28 in let-7 miRNA biogenesis will be investigated by in vivo and in vitro RNA binding assays. A sensitive RNAi based screen will be undertaken to identify both enhancers and suppressors of lin-28 mutant phenotypes that are dependent on let-7 activity. (B) A novel function for the Period homolog, LIN-42, in regulating miRNA maturation will be investigated by determining RNA targets and genetic interactors. 3. Identify in vivo miRNA target sites bound by ALG-1 and the regulatory outcome of the interactions. (A) Sequences bound and regulated by ALG-1 that are dependent on specific miRNAs will be identified using a novel biochemical and computational approach combined with microarray analyses. (B) Specific miRNA targets will be validated by high throughput genetic approaches and sensitive reporter gene assays. PUBLIC HEALTH RELEVANCE: Normal cells become cancerous when gene regulatory pathways are disrupted. MicroRNAs comprise a new class of gene regulatory molecules, and aberrations in the expression and function of specific microRNAs contribute to oncogenesis. By studying the essential role of microRNAs in normal cellular development, this proposal aims to help elucidate how disruption of this pathway can lead to cancerous states.