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

项目摘要 遗传调控是所有发育和生理过程的基本组成部分。相反地， 疾病状态通常是基因功能被破坏或异常的有害后果。微rna 微RNA（miRNA）是普遍存在的小RNA，其构成了在细胞中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、胰腺的发育缺陷， 肠、肾、肝、乳腺、睾丸和卵巢，伴有生理和行为缺陷， 疾病如癫痫、耳聋、失明、不育、自身免疫、神经变性、糖尿病、心脏病 失败和癌症。在分子水平上，miRNAs作为Argonaute（AGO）蛋白的向导， 使用编码的序列信息来鉴定靶向抑制的信使RNA（mRNA）。因此，在本发明中， 对miRNA-靶相互作用的详细了解有可能阐明基因调控网络 对数百种发育、生理和疾病过程至关重要。尽管这个非凡的 潜在的、准确的miRNA靶点预测仍然是一个突出的挑战， 捕获miRNA-靶标相互作用是昂贵且困难的，限制了它们到专门的研究组。因此，在本发明中， 尽管深入了解哺乳动物生物学和发现新的药物靶点有巨大的机会， 哺乳动物中绝大多数必需的miRNA-靶标相互作用仍然未知。的目的 一项建议是创建和提供小鼠模型，用于稳健和可靠的miRNA靶点发现， 足以被不同的研究人员广泛使用。这一目标将通过以下两项具体工作来实现： 目的：1）确定在小鼠中标记AGO 2的最佳策略; 2）开发条件性AGO 2的小鼠模型。 发现miRNA靶点。在目标1下，我们将采用最近开发的方法，这些方法采用超稳定的 共价键从复杂的分子混合物中分离AGO-RNA加合物，用于发现AGO- RNA相互作用小鼠。我们将利用我们对AGO结构的了解，制定创新战略， 编码允许共价捕获AGO而不损害AGO功能或小鼠发育的标签。 在目标2下，我们将开发有条件表达标记形式的AGO的小鼠品系，其可用于 在特定细胞类型中发现miRNA靶点。这将是一个重大的进步，因为它将允许 研究人员分离参与离散生理过程和罕见细胞类型的miRNA靶点， 目前是不可能的。我们的理由是，强大、可靠和可访问的工具将允许不同的研究人员 发现与几乎所有NIH研究所相关的过程和疾病中的miRNA-靶标相互作用， 中心，以及大大推进了解这些无处不在的调节分子的生物学。

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.