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: 8208166
• 负责人: Hilary A Coller
• 金额: $ 4.51万
• 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 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8208166
• 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; Neurodegenerative Disorders; 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; research study; web site

SUMMARY/ABSTRACT 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. PROJECT NARRATIVE 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.

摘要/摘要 microRNA（miRNA）是各种生物过程的中心调节剂，包括癌症， 发育，神经退行性和代谢性疾病以及病毒感染。但是，他们的分子细节 机制仍然存在争议。在人类细胞中细胞静止的研究过程中，我们 发现Let-7 miRNA可能不仅调节具有识别序列的3'UTR中的基因，还可以调节 还具有识别序列在其编码区域中的基因。我们进一步发现了证据 3'UTR与编码区域识别位点的靶向之间的机械差异。我们的结果 结合发布的数据还提高了miRNA机制中目标特异性的可能性 静止的上下文。我们在这里提出了一种合并的计算和实验方法，以测试我们的 假设miRNA具有不同的作用机理，具体取决于 miRNA和目标。我们预计结果对于预测生理相关目标是有价值的 miRNA和设计最大有效的miRNA疗法。 在我们的第一个目标中，我们将使用比较基因组学和记者测定法的组合来确定是否是否 有多种不同的机制，miRNA靶向不同部分的转录本。具体 目的＃2-＃4，我们将测试我们的假设，即有一些特定于成绩单的机制来控制是否是否存在 miRNA通过转录本降解或翻译调节靶标。我们将分析用一种转染的细胞 通过对总转录本和多元素相关的转录本进行微阵列分析，通过进行微阵列分析。 这些数据将使我们能够确定miRNA-target相互作用的特征，从而导致成绩单 降解和影响翻译启动的降解。在特定的目标＃3中，我们将将这些实验扩展到 测试我们的假设，即miRNA活性从抑制到激活时有一个特异性转换 变得静止。最后，在特定的目标＃4中，我们将通过 监测抗MIR，miRNA抑制剂对总和相关转录物的影响。我们的 期望是对miRNA功能的机制解密，包括特异性的基础 miRNA-target相互作用将为miRNA研究领域提供清晰度。调查结果将做出 通过我们的网站获得，随着研究人员研究各种各样的研究人员，还将推进许多相关领域 生物过程和疾病状态可以更好地将miRNA与其目标和功能联系起来。项目叙述 尽管miRNA显然是包括癌症在内的各种生物学过程中的核心参与者，但 发育，神经退行性和代谢性疾病以及病毒感染，将miRNA与特定 它们调节的分子一直是一个挑战。我们在这里提出了一个组合的计算和 实验方法来阐明许多不同miRNA- 目标配对。我们预计这些发现对广泛领域的研究人员具有很高的价值 努力识别miRNA靶标，以及将miRNA设计为治疗剂的靶标。