Combinatorial microRNA regulation of cardiac transcription factors

心脏转录因子的组合 microRNA 调节

• 批准号: 8218634
• 负责人: RICHARD H. GOODMAN
• 金额: $ 23.1万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-12-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8218634
• 关键词: 3' Untranslated Regions; Affect; Algorithms; Binding; Bioinformatics; Biological; Biological Assay; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cells; Characteristics; Complex; Data; Dominant-Negative Mutation; Elements; Goals; Heart; Heart Diseases; Human; Individual; Mediating; Messenger RNA; Methods; MicroRNAs; Modeling; Monitor; Mutate; Mutation; Pathway interactions; Proteins; RNA-Induced Silencing Complex; Regulation; Reporter; Repression; Role; Seminal; Signal Transduction; Small RNA; Specificity; System; Testing; Tissues; Untranslated Regions; base; combinatorial; design; inhibitor/antagonist; insight; novel; novel strategies; prevent; ratiometric; sensor; transcription factor

DESCRIPTION (provided by applicant): The fact that individual cardiac cells can express hundreds of microRNAs, each with potentially hundreds of targets, raises the question of how such a complex mode of regulation can possibly achieve specificity. We hypothesize that this occurs through the ability of individual 3'UTRs to interact with multiple microRNAs simultaneously, such that concurrent binding of two (or more) microRNAs is required for biologically meaningful regulation. Using a method that we developed to identify microRNA-mRNA interactions biochemically, we showed that miR-1 and miR-133a associate simultaneously with the 3'UTR of the cardiac transcription factor, Hand2. This is probably the best example to date in any system of concurrent binding by two microRNAs to a single target. We hypothesize that binding of miR-1 and miR-133a to the Hand2 3'UTR is mutually interdependent, such that both microRNAs must associate with their recognition elements (MREs) to achieve efficient repression. Such a mechanism would increase signaling complexity, generating specificity of targeting and constraining the number of effective mRNA targets for an individual microRNA. This scenario may be characteristic of other microRNA targets, and we will use a new assay developed in our lab to identify additional cardiac mRNAs that are subject to a similar dual mode of regulation. These studies could answer a seminal question in microRNA signaling-how the multitude of microRNAs and predicted targets can achieve specificity. To accomplish our goals, we will determine how miR-1 and miR-133a cooperate to regulate Hand2 expression. Our data indicates that binding of miR-1 and miR-133a is mutually interdependent, which has never before been described. We will elucidate the functional implications of this finding by utilizing a set of novel bidirectional ratiometric sensors. To elucidate the mechanism underlying the interdependency of miR-1 and miR-133a binding, we will target Ago2, a core component of the RNA induced silencing complex (RISC), to the mutated Hand2 miR-1 or miR-133a MREs to determine whether recruitment of Ago2 and associated proteins can rescue effects of the MRE mutations. We will also determine whether the coordinate regulation by miR-1 and miR-133a is shared by other cardiac mRNAs. Bioinformatic algorithms designed to predict microRNA targets are notoriously imprecise in their ability to identify authentic interactions, missing many interactions and falsely predicting others. We developed a novel approach for identifying these targets that employs a dominant negative RISC component to trap microRNA-mRNA intermediates prior to degradation. We will use this approach to identify other cardiac mRNAs that associate with both miR-1 and miR-133a and test whether microRNA binding and function are similarly interdependent. Understanding how combinations of microRNAs affect expression of targets is essential for developing effective microRNA-based therapies. PUBLIC HEALTH RELEVANCE: This project uses several novel approaches to examine the role of micro RNA pathways in regulating cardiac transcription factors with implications for human congenital and acquired heart disease. Characterization of these pathways may identify new therapies for treating cardiovascular disease.

描述(由申请人提供)：单个心肌细胞可以表达数百个microRNAs，每个细胞可能有数百个靶点，这引发了这样一个复杂的调控模式如何可能实现特异性的问题。我们假设这是通过单个3‘UTRs同时与多个microRNAs相互作用来实现的，因此需要同时结合两个(或更多)microRNAs才能进行有生物学意义的调控。使用我们开发的一种生化方法来鉴定microRNA-mRNA的相互作用，我们发现miR-1和miR-133a同时与心脏转录因子Hand2的3‘UTR区相关。这可能是迄今为止由两个microRNAs同时结合到单个靶标的任何系统中的最好例子。我们假设miR-1和miR-133a与Hand2 3‘UTR的结合是相互依赖的，因此两个microRNAs必须与它们的识别元件(MRE)结合才能实现有效的抑制。这种机制会增加信号的复杂性，产生靶向的特异性，并限制单个microRNA的有效信使核糖核酸靶标的数量。这种情况可能是其他microRNA靶标的特征，我们将使用我们实验室开发的一种新的分析方法来识别其他受到类似双重调节模式的心脏mRNAs。这些研究可以回答microRNA信号传递中的一个开创性问题--众多的microRNA和预测的靶标如何实现特异性。为了实现我们的目标，我们将确定miR-1和miR-133a如何合作调控Hand2的表达。我们的数据表明miR-1和miR-133a的结合是相互依赖的，这在以前从未被描述过。我们将利用一组新的双向比率传感器来阐明这一发现的功能含义。为了阐明miR-1和miR-133a结合相互依赖的机制，我们将RNA诱导沉默复合体(RISC)的核心成分Ago2靶向突变的Hand2、miR-1或miR-133a MRES，以确定Ago2及其相关蛋白的招募是否可以挽救MRE突变的影响。我们还将确定miR-1和miR-133a的协调调节是否与其他心脏mRNAs共享。为预测microRNA靶标而设计的生物信息学算法在识别真实相互作用、遗漏许多相互作用和错误预测其他相互作用方面的能力是出了名的不准确。我们开发了一种新的方法来识别这些靶标，该方法利用一个显性的负RISC成分在降解之前捕获microRNA-mRNA中间体。我们将使用这种方法来识别与miR-1和miR-133a相关的其他心脏mRNAs，并测试microRNA结合和功能是否类似地相互依赖。了解microRNA的组合如何影响靶标的表达，对于开发有效的基于microRNA的疗法至关重要。 公共卫生相关性：该项目使用几种新的方法来研究微小RNA途径在调节心脏转录因子中的作用，这与人类先天性和获得性心脏病有关。对这些途径的表征可能会确定治疗心血管疾病的新疗法。