A Combined Computational and Experimental Approach to Defining Mechanisms of micr

定义微观机制的计算和实验相结合的方法

• 批准号: 7749017
• 负责人: Hilary A Coller
• 金额: $ 28.47万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7749017
• 关键词: 3' Untranslated Regions; Affect; Animals; Behavior; Binding; Binding Sites; Biogenesis; Biological Assay; Biological Process; Cell Cycle Arrest; Cells; Characteristics; Code; Data; Development; Disease; Exhibits; Gene Expression; Genes; Genome; Human; Immunoblotting; Individual; Link; Malignant Neoplasms; Mediating; Metabolic Diseases; MicroRNAs; Microarray Analysis; Molecular; Monitor; Nerve Degeneration; Pattern; Physiological; Play; Polyribosomes; Process; Proliferating; Proteins; Publishing; Regulation; Reporter; Reporting; Repression; Research; Research Personnel; Role; Sequence Alignment; Site; Specificity; Testing; Therapeutic; Time; Transcript; Translating; Translation Initiation; Translations; Untranslated Regions; Virus Diseases; Work; base; comparative genomics; design; expectation; genome-wide; human DICER1 protein; inhibitor/antagonist; insight; public health relevance; research study; web site

DESCRIPTION (provided by applicant): microRNAs (miRNAs) are central regulators of a wide array of biological processes including cancer, development, neurodegenerative and metabolic diseases and viral infection. Yet, the details of their molecular mechanisms remain controversial. In the course of our studies on cellular quiescence in human cells, we discovered that the let-7 miRNA likely regulates not only genes with recognition sequences in their 3' UTRs but also genes with recognition sequences in their coding regions. We further discovered evidence for mechanistic differences between the targeting of 3' UTR versus coding region recognition sites. Our results in combination with published data also raise the possibility of target-specificity in miRNA mechanisms in the context of quiescence. We propose here a combined computational and experimental approach to test our hypothesis that miRNAs have distinct mechanisms of action depending upon the interaction between the miRNA and the target. We expect that the results will be valuable for predicting physiologically relevant targets of miRNAs and for designing maximally effective miRNA therapeutics. In our first aim, we will use a combination of comparative genomics and reporter assays to determine whether there are multiple distinct mechanisms by which miRNAs target different portions of transcripts. In Specific aims #2 - #4, we will test our hypothesis that there are transcript-specific mechanisms that govern whether miRNAs regulate targets via transcript degradation or translation. We will analyze cells transfected with one of several miRNAs by performing microarray analysis on total transcripts and polysome-associated transcripts. These data will allow us to identify characteristics of miRNA-target interactions that result in transcript degradation and those that affect translation initiation. In specific aim #3, we will extend these experiments to test our hypothesis that there is a target-specific switch in miRNA activity from repression to activation as cells become quiescent. Finally, in specific aim #4, we will extend our analysis to endogenous miRNAs by monitoring the impact of anti-miRs, miRNA inhibitors, on total and polysome-associated transcripts. Our expectation is that deciphering the mechanisms of miRNA function, including the basis for specificity for miRNA-target interactions, will provide clarity to the field of miRNA research. The findings, which will be made available through our website, will also advance many related fields as researchers studying a wide array of biological processes and disease states are better able to associate miRNAs with their targets and functions. PUBLIC HEALTH RELEVANCE: While miRNAs are clearly central players in a wide array of biological processes including cancer, development, neurodegenerative and metabolic diseases and viral infection, linking miRNAs with the specific molecules that they regulate has been a challenge. We propose here a combined computational and experimental approach to elucidate the mechanisms of miRNA-target interactions for many different miRNA- target pairings. We anticipate that these findings will be of great value to researchers in a wide range of fields working to identify miRNAs targets, and those designing miRNAs as therapeutics.

描述（由申请人提供）：microRNA (miRNA) 是多种生物过程的中心调节因子，包括癌症、发育、神经退行性和代谢疾病以及病毒感染。然而，其分子机制的细节仍然存在争议。在我们对人类细胞的细胞静止的研究过程中，我们发现let-7 miRNA可能不仅调节其3'UTR中具有识别序列的基因，而且还调节其编码区中具有识别序列的基因。我们进一步发现了 3' UTR 靶向与编码区识别位点之间机制差异的证据。我们的结果与已发表的数据相结合也提出了在静止背景下 miRNA 机制的靶点特异性的可能性。我们在这里提出了一种组合的计算和实验方法来检验我们的假设，即 miRNA 具有不同的作用机制，具体取决于 miRNA 和靶标之间的相互作用。我们期望这些结果对于预测 miRNA 的生理相关靶点和设计最有效的 miRNA 疗法具有重要价值。 在我们的第一个目标中，我们将结合使用比较基因组学和报告基因检测来确定 miRNA 是否有多种不同的机制靶向转录本的不同部分。在特定目标 #2 - #4 中，我们将测试我们的假设，即存在转录特异性机制来控制 miRNA 是否通过转录降解或翻译来调节靶标。我们将通过对总转录本和多核糖体相关转录本进行微阵列分析来分析用几种 miRNA 之一转染的细胞。这些数据将使我们能够识别导致转录本降解和影响翻译起始的 miRNA-靶标相互作用的特征。在具体目标#3中，我们将扩展这些实验来检验我们的假设，即当细胞变得静止时，miRNA 活性存在从抑制到激活的目标特异性转变。最后，在具体目标 4 中，我们将通过监测抗 miR、miRNA 抑制剂对总转录本和多核糖体相关转录本的影响，将我们的分析扩展到内源 miRNA。我们期望破译 miRNA 功能机制，包括 miRNA-靶标相互作用的特异性基础，将为 miRNA 研究领域提供清晰的思路。这些研究结果将通过我们的网站公布，也将推动许多相关领域的发展，因为研究各种生物过程和疾病状态的研究人员能够更好地将 miRNA 与其目标和功能联系起来。公共健康相关性：虽然 miRNA 显然是癌症、发育、神经退行性和代谢疾病以及病毒感染等一系列生物过程的核心参与者，但将 miRNA 与其调节的特定分子联系起来一直是一个挑战。我们在这里提出了一种组合的计算和实验方法来阐明许多不同 miRNA-靶标配对的 miRNA-靶标相互作用的机制。我们预计这些发现将对各个领域致力于识别 miRNA 靶标以及设计 miRNA 作为治疗药物的研究人员具有巨大价值。