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）是各种生物学过程的中心调节剂，包括癌症，发育，神经退行性和代谢性疾病以及病毒感染。然而，它们的分子机制的细节仍然存在争议。在我们在人类细胞中细胞静止的研究过程中，我们发现Let-7 miRNA不仅可以调节其3'UTR中具有识别序列的基因，而且还调节具有识别序列的基因在编码区域中。我们进一步发现了3'UTR与编码区域识别位点的靶向靶向之间的机理差异的证据。我们的结果与已发布的数据结合使用，还增加了在静止下的miRNA机制中目标特异性的可能性。我们在这里提出了一种合并的计算和实验方法，以检验我们的假设，即miRNA具有不同的作用机理，具体取决于miRNA与目标之间的相互作用。我们预计结果对于预测miRNA的生理相关靶标和设计最大有效的miRNA疗法将是有价值的。 在我们的第一个目标中，我们将使用比较基因组学和报告基因测定法的组合来确定miRNA是否有多种不同的机制靶向不同部分的转录本。在特定的目标＃2-＃4中，我们将检验我们的假设，即有一些转录本特异性机制来控制miRNA是否通过转录本降解或翻译调节靶标。我们将通过对总转录本和与多聚糖相关的转录本进行微阵列分析来分析用几个miRNA转染的细胞。这些数据将使我们能够确定miRNA-target相互作用的特征，从而导致转录本降解和影响翻译起始的特征。在特定的目标＃3中，我们将扩展这些实验，以测试我们的假设，即随着细胞变得静止，miRNA活性从抑制到激活存在靶标特异性。最后，在特定的目标＃4中，我们将通过监测抗MIR，miRNA抑制剂对总和多溶血体相关转录本的影响，将分析扩展到内源miRNA。我们的期望是，解密miRNA功能的机制，包括miRNA-target相互作用的特异性基础，将为miRNA研究领域提供清晰度。这些发现将通过我们的网站提供，还将推进许多相关领域，因为研究各种生物过程和疾病状态的研究人员可以更好地将miRNA与其目标和功能联系起来。公共卫生相关性：尽管MiRNA显然是众多生物学过程中的主要参与者，包括癌症，发育，神经退行性和代谢疾病以及病毒感染，将miRNA与调节的特定分子联系起来是一个挑战。我们在这里提出了一种合并的计算和实验方法，以阐明许多不同miRNA目标配对的miRNA-artet art靶相互作用的机制。我们预计，这些发现对于在广泛的领域的研究人员而言，这些发现将具有巨大的价值，这些领域旨在识别miRNA目标，以及将miRNA作为治疗学设计的靶标。