Combinatorial microRNA regulation of cardiac transcription factors

心脏转录因子的组合 microRNA 调节

• 批准号: 8392248
• 负责人: RICHARD H. GOODMAN
• 金额: $ 18.33万
• 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/8392248
• 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.

描述（由申请人提供）：单个心脏细胞可以表达数百种microRNA，每种microRNA都可能具有数百种靶点，这一事实提出了这样一种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 'UTR同时与多个microRNA相互作用的能力发生的，因此需要两个（或更多个）microRNA的同时结合才能进行有生物学意义的调控。使用我们开发的用于鉴定microRNA-mRNA相互作用的生物化学方法，我们发现miR-1和miR-133 a同时与心脏转录因子Hand 2的3 'UTR结合。这可能是迄今为止在任何两个microRNA同时结合到单个靶标的系统中最好的例子。我们假设miR-1和miR-133 a与Hand 2 3 'UTR的结合是相互依赖的，因此这两种microRNA必须与它们的识别元件（MRE）结合以实现有效的抑制。这种机制将增加信号传导的复杂性，产生靶向的特异性，并限制单个microRNA的有效mRNA靶点的数量。这种情况可能是其他microRNA靶点的特征，我们将使用我们实验室开发的一种新的检测方法来鉴定其他受到类似双重调控模式影响的心脏mRNA。这些研究可以回答microRNA信号中的一个开创性问题-大量microRNA和预测的靶点如何实现特异性。为了实现我们的目标，我们将确定miR-1和miR-133 a如何合作调节Hand 2表达。我们的数据表明，miR-1和miR-133 a的结合是相互依赖的，这是以前从未描述过的。我们将阐明这一发现的功能的影响，利用一组新的双向比率传感器。为了阐明miR-1和miR-133 a结合相互依赖的机制，我们将Ago 2（RNA诱导沉默复合物（RISC）的核心组分）靶向突变的Hand 2 miR-1或miR-133 a MRE，以确定Ago 2和相关蛋白的募集是否可以挽救MRE突变的效应。我们还将确定miR-1和miR-133 a的协调调节是否被其他心脏mRNA共享。众所周知，旨在预测微小RNA靶点的生物信息学算法在识别真实相互作用的能力方面不精确，错过了许多相互作用并错误地预测了其他相互作用。我们开发了一种新的方法来鉴定这些靶标，该方法采用显性负RISC组分在降解前捕获microRNA-mRNA中间体。我们将使用这种方法来鉴定与miR-1和miR-133 a相关的其他心脏mRNA，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。