Molecular recognition and regulation in microRNA processing by the DGCR8 protein

DGCR8 蛋白对 microRNA 加工的分子识别和调控

• 批准号: 7628447
• 负责人: Feng Guo
• 金额: $ 26.33万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-10 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7628447
• 关键词: Address; Amino Acids; Belief; Binding; Biochemical; Biological; Biological Assay; Biological Process; Cell Culture Techniques; Cell Nucleus; Cells; Cleaved cell; Code; Complex; Cultured Cells; Cytoplasm; Data; Development; DiGeorge Syndrome; Disease; Dominant-Negative Mutation; Eukaryotic Cell; Functional RNA; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genomics; Goals; Heme; Human; Human Genome; In Vitro; Knowledge; Learning; Left; Link; Medical; Messenger RNA; Methods; MicroRNAs; Microprocessor; Modeling; Molecular; Molecular Conformation; Mutagenesis; Mutation; Pathologic Processes; Patients; Plasmids; Process; Proteins; RNA; RNA Binding; RNA-Binding Proteins; Regulation; Reporting; Research; Research Personnel; Ribonucleases; Roentgen Rays; Screening procedure; Site-Directed Mutagenesis; Structure; Testing; Therapeutic; Therapeutic Agents; Transcript; Translational Repression; Tumor Suppressor Proteins; X-Ray Crystallography; base; cancer cell; cofactor; combinatorial; design; heme a; heme-binding protein; improved; in vitro Assay; in vivo; knock-down; molecular recognition; mutant; neoplastic cell; novel; overexpression; pri-miRNA; programs; research study; three dimensional structure

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) have been recognized as an important class of non-coding RNAs that are involved in a variety of biological and pathological processes. The broad, long-term objectives are to elucidate the molecular mechanism through which long miRNA primary transcripts (pri-miRNAs) are specifically recognized by cellular factors for processing and how their maturation is regulated through a mechanism involving heme. Specifically, we will focus on the structure and function of a key factor-an human RNA binding protein called DGCR8 that is heterozygously deleted in DiGeorge syndrome patients. We expect to fill major gaps in our current knowledge of miRNAs: some critical features of miRNA genes have not been identified so that miRNA genes cannot be accurately predicted from genomic sequences; and little is known about how the processing of miRNAs are regulated. Specific aim 1 will elucidate how DGCR8 interacts with pri-miRNAs. The protein-RNA interface will be examined using biochemical and X-ray crystallographic methods. The origin of the binding cooperativity will be investigated using mutagenesis methods. In specific aim 2, the interactions between DGCR8 and the heme cofactor will be revealed. The amino acid residues important for heme binding will be identified using site-directed mutagenesis and screening methods. The structure of the heme-binding domain will be determined using X-ray crystallography or NMR. The molecular mechanism of autoinhibition of DGCR8 will be examined using in vitro methods. In specific aim 3, the importance of the heme-DGCR8 interaction and the autoinhibition of DGCR8 in pri-miRNA processing will be investigated in human cell cultures. Medical revelence: MicroRNAs are a new class of genes that are involved in important biological and disease processes. Our research aims to improve our abilities to find these genes in the human genome, to develop better theraprutic microRNAs that can modulate abnormal gene expression in disease states. Our discovery that heme may be involved in regulating the processing of microRNAs suggest that they can be used as therapeutic agents for microRNA-related diseases.

描述（由申请人提供）：MicroRNA（miRNA）已被认为是一类重要的非编码RNA，参与多种生物和病理过程。广泛的长期目标是阐明长 miRNA 初级转录物 (pri-miRNA) 被细胞因子特异性识别进行加工的分子机制，以及如何通过涉及血红素的机制来调节它们的成熟。具体来说，我们将重点关注一个关键因素的结构和功能，即一种名为 DGCR8 的人类 RNA 结合蛋白，该蛋白在迪乔治综合征患者中杂合缺失。我们期望填补我们目前对 miRNA 知识的主要空白：miRNA 基因的一些关键特征尚未确定，因此无法从基因组序列准确预测 miRNA 基因；对于 miRNA 的加工是如何调控的，人们知之甚少。具体目标 1 将阐明 DGCR8 如何与 pri-miRNA 相互作用。将使用生化和 X 射线晶体学方法检查蛋白质-RNA 界面。将使用诱变方法研究结合协同性的起源。在具体目标 2 中，将揭示 DGCR8 和血红素辅因子之间的相互作用。对于血红素结合重要的氨基酸残基将使用定点诱变和筛选方法来鉴定。血红素结合域的结构将使用 X 射线晶体学或 NMR 确定。将使用体外方法检查 DGCR8 自抑制的分子机制。在具体目标 3 中，将在人类细胞培养物中研究血红素-DGCR8 相互作用的重要性以及 pri-miRNA 加工中 DGCR8 的自身抑制。医学意义：MicroRNA 是一类参与重要生物和疾病过程的新型基因。我们的研究旨在提高我们在人类基因组中寻找这些基因的能力，开发更好的治疗性 microRNA，以调节疾病状态下的异常基因表达。我们发现血红素可能参与调节 microRNA 的加工，这表明它们可以用作 microRNA 相关疾病的治疗剂。