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

秀丽隐杆线虫let-7 microRNA生物发生和功能的调控

• 批准号: 8411987
• 负责人: AMY E. PASQUINELLI
• 金额: $ 28.23万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8411987
• 关键词: Address; Affect; Amino Acid Sequence; Binding; Binding Sites; Biochemical; Biogenesis; Biological Assay; Caenorhabditis elegans; Cancerous; Candidate Disease Gene; Circadian Rhythms; Cloning; Co-Immunoprecipitations; Code; Complex; Development; Disease; Elements; Enhancers; Family; Functional RNA; Gene Expression; Gene Targeting; Genes; Genetic; Health; Homologous Gene; Human; Immunoprecipitation; In Vitro; Individual; Knowledge; Lead; Link; Lobular Neoplasia; Mediating; Methods; MicroRNAs; Modeling; Molecular; Nematoda; Normal Cell; Outcome; Pathway interactions; Peptide Sequence Determination; Phenotype; Population; Post-Transcriptional Regulation; Process; Protein Microarray Assay; Proteins; RNA; RNA Binding; RNA Interference; Regulation; Regulator Genes; Regulatory Pathway; Reporter; Reporter Genes; Research; Role; Site; Small RNA; Structure; Testing; Trans-Splicing; Transcript; Tumor Suppressor Proteins; Up-Regulation; base; cancer cell; cellular development; cis acting element; crosslink; deep sequencing; design; human disease; in vivo; mRNA Expression; member; mutant; novel; programs; public health relevance; tumorigenesis

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.

描述（由申请人提供）：微小RNA（miRNAs）包括一类新发现的非编码RNA基因，特定的miRNAs已经与人类健康和疾病相关的关键基因调控作用相关。let-7基因是这个新的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在物种间非常保守，其作为人类肿瘤抑制因子的潜在作用与其促进细胞分化的功能有关，最初在C.优雅的本更新中描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鉴定以及我们开发的一种稳健的生物化学方法的基础上，该方法旨在揭示包括argonaute蛋白ALG-1在内的miRNA复合物如何识别和调节体内直接靶基因。我们的研究对于理解miRNAs如何在物种间表达和发挥作用以及这些途径的干扰如何导致人类疾病具有广泛的相关性。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，我们提出了以下目标。1.描述控制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使用体外结构分析和体内表达和功能测定来确定。(B)将定义介导let-7相关miRNA（mir-84、mir-48、mir-241和mir-795）的转录后调节的主要转录物和元件。2.确定LIN-28和LIN-42蛋白在调节miRNA表达中的功能。(A)lin-28在let-7 miRNA生物合成中的作用将通过体内和体外RNA结合测定来研究。将进行基于灵敏RNAi的筛选以鉴定依赖于let-7活性的lin-28突变表型的增强子和抑制子。(B)一个新的功能周期同源物，LIN-42，在调节miRNA的成熟将通过确定RNA的目标和遗传相互作用。3.鉴定ALG-1结合的体内miRNA靶位点和相互作用的调控结果。(A)ALG-1依赖于特定的miRNAs的结合和调控序列将使用一种新的生物化学和计算方法结合微阵列分析进行鉴定。(B)将通过高通量遗传学方法和敏感的报告基因测定来验证特异性miRNA靶点。