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

用于解码不同组织和细胞类型中 microRNA 调控的小鼠模型

基本信息

批准号：
10194631
负责人：
IAN JOHN MACRAE
金额：
$ 22.19万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-15 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10194631
关键词：
Animals Autoimmunity Automobile Driving Behavioral Binding Biochemical Biological Biological Process Biology Blindness Brain Breast Cell physiology Cells Complex Cues Defect Development Developmental Process Diabetes Mellitus Diagnosis Disease Disease model Ear Epilepsy Eye Family Funding Gene Expression Regulation Genes Genetic Goals Health Heart Heart failure Human Infertility Institutes Intestines Kidney Knowledge Liver Lung Malignant Neoplasms Mammals Messenger RNA Methods MicroRNAs Molecular Mus Muscle Nerve Degeneration Ovary Pancreas Pathway interactions Phenotype Physiological Physiological Processes Play Post-Transcriptional Regulation Prevention Process Production Protein Engineering Proteins RNA Regulation Regulator Genes Repression Research Research Personnel Role Site Skeleton Small RNA Specific qualifier value Structure System Techniques Technology Testis Tissues Tooth structure United States National Institutes of Health Work adduct cell type cost covalent bond deafness developmental disease gene function human disease in vivo innovation insight mouse development mouse model new therapeutic target novel prevent small molecule tool transcriptome

项目摘要

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.

项目概要遗传调控是所有发育和生理过程的基本组成部分。反过来，疾病状态通常是基因功能破坏或异常的有害后果。微小RNA (miRNA) 是普遍存在的小 RNA，构成了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、卵巢，伴有生理和行为缺陷，癫痫、耳聋、失明、不孕症、自身免疫、神经退行性疾病、糖尿病、心脏病等疾病失败和癌症。在分子水平上，miRNA 充当 Argonaute (AGO) 蛋白的向导，该蛋白使用编码的序列信息来识别用于抑制的信使 RNA (mRNA)。因此， miRNA-靶标相互作用的详细知识有可能阐明遗传调控网络对于数百种发育、生理和疾病过程至关重要。尽管有如此非凡的潜在的、准确的 miRNA 靶点预测仍然是一个突出的挑战，并且生化方法捕获 miRNA 与靶点之间的相互作用既昂贵又困难，因此仅限于专门的研究小组。因此，尽管有巨大的机会深入了解哺乳动物生物学和发现新的药物靶标，哺乳动物中绝大多数重要的 miRNA-靶标相互作用仍然未知。此举的目的提议是创建并提供小鼠模型，以便轻松可靠地发现 miRNA 靶点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标：1) 确定在小鼠中标记 AGO2 的最佳策略； 2) 开发条件小鼠模型 miRNA 靶标发现。在目标 1 下，我们将采用最近开发的方法，该方法采用超稳定共价连接从复杂的分子混合物中分离 AGO-RNA 加合物，以发现 AGO- RNA相互作用小鼠。我们将利用我们对 AGO 结构的了解来制定创新策略编码标签允许共价捕获 AGO，而不影响 AGO 功能或小鼠发育。在目标 2 下，我们将开发有条件表达 AGO 标记形式的小鼠品系，可用于特定细胞类型中 miRNA 靶标的发现。这将是一个重大进步，因为它将允许研究人员分离出参与离散生理过程和稀有细胞类型的 miRNA 靶标，目前不可能。我们的理由是，强大、可靠且易于使用的工具将使不同的研究人员能够发现与几乎所有 NIH 机构相关的过程和疾病中 miRNA 靶点相互作用中心，并极大地促进对这些无处不在的调节分子的生物学的理解。