Mouse models for decoding microRNA regulation in diverse tissues and cell types

项目总结 基因调控是所有发育和生理过程的基本组成部分。相反， 疾病状态往往是基因功能中断或异常的有害后果。MicroRNAs (MiRNA)是普遍存在的小RNA，构成了体内转录后基因调控的主要水平 动物。事实上，破坏人类和小鼠之间保守的miRNA基因会导致不同的表型， 包括大脑、眼睛、耳朵、心脏、肺、肌肉、骨骼、牙齿、胰腺、 肠道、肾脏、肝脏、乳房、睾丸和卵巢，伴有生理和行为缺陷 癫痫、耳聋、失明、不孕不育、自身免疫、神经变性、糖尿病、心脏等疾病 失败和癌症。在分子水平上，miRNAs作为ArgAerte(AGO)蛋白质的向导，它 使用编码的序列信息来识别作为抑制目标的信使RNA(MRNA)。因此， 对miRNA-靶相互作用的详细了解有可能阐明遗传调控网络。 对于成百上千的发育、生理和疾病过程是必不可少的。尽管这是非同寻常的 潜在的、准确的miRNA靶标预测仍然是一个突出的挑战，而生化方法 捕获miRNA与靶标的相互作用既昂贵又困难，仅限于专门的研究小组。因此， 尽管有深入了解哺乳动物生物学和发现新的药物靶点的巨大机会，但 哺乳动物中绝大多数重要的miRNA-靶相互作用仍不清楚。这样做的目的是 建议是创建和提供小鼠模型，以便轻松、可靠地发现miRNA靶标 足以被不同的研究人员广泛使用。这一目标将通过追求两个具体目标来实现 目的：1)确定标记小鼠AGO2的最佳策略；2)建立条件性条件下的小鼠模型 MiRNA-目标发现。在目标1下，我们将采用最近开发的方法，这些方法使用超稳定 从复杂分子混合物中分离AgO-RNA加合物的共价键，用于发现AgO-RNA加合物 小鼠的RNA相互作用。我们将利用我们对AGO结构的了解来制定创新的战略 编码标签，允许共价捕获AGO，而不影响AGO功能或鼠标开发。 在目标2下，我们将开发有条件地表达标记形式的AGO的鼠系，可用于 MiRNA-在指定的细胞类型中发现目标。这将是一个重大的进步，因为它将使 研究人员分离涉及离散生理过程和稀有细胞类型的miRNA靶标， 目前是不可能的。我们的理性是，健壮、可靠和可访问的工具将允许不同的研究人员 要发现与几乎所有NIH研究所和疾病相关的过程和疾病中的miRNA-靶相互作用 中心，以及极大地促进了对这些无处不在的调节分子的生物学的理解。

PROJECT SUMMARY Genetic regulation is a fundamental component of all developmental and physiological processes. Conversely, disease states are very often the harmful consequences of disrupted or aberrant gene function. MicroRNAs (miRNA) are ubiquitous small RNAs that constitute a major level of post-transcriptional gene regulation in animals. Indeed, disrupting miRNA genes conserved between humans and mice leads to diverse phenotypes, including defects in the development of the brain, eyes, ears, heart, lungs, muscle, skeleton, teeth, pancreas, intestine, kidneys, liver, breast, testes, and ovaries, accompanied by physiological and behavioral defects and diseases such as epilepsy, deafness, blindness, infertility, autoimmunity, neurodegeneration, diabetes, heart failure, and cancer. On the molecular level, miRNAs function as guides for Argonaute (AGO) proteins, which use the encoded sequence information to identify messenger RNAs (mRNA) targeted for repression. Thus, detailed knowledge of miRNA-target interactions has the potential to illuminate genetic regulatory networks essential to hundreds of developmental, physiological, and disease processes. Despite this extraordinary potential, accurate miRNA-target prediction remains an outstanding challenge, and biochemical methods for capturing miRNA-target interactions are costly and difficult, limiting them to specialized research groups. Thus, despite the enormous opportunity for insight into mammalian biology and discovery of novel drug targets, the vast majority of essential miRNA-target interactions in mammals remain unknown. The objective of this proposal is to create and provide mouse models for robust and reliable miRNA-target discovery that are easy enough to be widely used by diverse researchers. This objective will be achieved by pursuing two specific aims: 1) Identify optimal strategies for tagging AGO2 in mice; and, 2) Develop mouse models for conditional miRNA-target discovery. Under Aim 1 we will adapt recently developed methods, which employ ultra-stable covalent linkages to isolate AGO-RNA adducts from complex molecular mixtures, for the discovery of AGO- RNA interactions mice. We will use our knowledge of AGO structure to devise innovative strategies for encoding tags that allow covalent capture of AGO without compromising AGO function or mouse development. Under Aim 2 we will develop mouse lines that conditionally express tagged forms of AGO that can be used for miRNA-target discovery in specified cell types. This will be a significant advance because it will allow researchers to isolate miRNA-targets involved in discrete physiological processes, and in rare cell types, which is currently not possible. Our rational is that robust, reliable, and accessible tools will allow diverse researchers to discover miRNA-target interactions in processes and diseases relevant to nearly all NIH institutes and centers, as well as greatly advance understanding the biology of these ubiquitous regulatory molecules.