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

基本信息

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
项目类别：
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

项目摘要

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）已被认为是参与多种生物学和病理过程的重要一类非编码RNA。广泛的长期目标是阐明长miRNA原发性转录本（PRI-MIRNA）的分子机制，通过细胞因子进行处理以及如何通过涉及血红素的机制调节其成熟。具体而言，我们将重点关注一个称为DGCR8的关键因子RNA结合蛋白的结构和功能，该蛋白在Digeorge综合征患者中被杂合删除。我们期望在当前对miRNA的知识中填补主要空白：尚未确定miRNA基因的某些关键特征，因此不能从基因组序列中准确预测miRNA基因。关于如何调节miRNA的处理，知之甚少。特定的目标1将阐明DGCR8如何与PRI-MIRNA相互作用。将使用生化和X射线晶体学方法对蛋白-RNA界面进行检查。结合协同的起源将使用诱变方法研究。在特定的目标2中，将揭示DGCR8与血红素辅因子之间的相互作用。将使用位置定向的诱变和筛选方法来鉴定对血红素结合重要的氨基酸残基。血红素结合域的结构将使用X射线晶体学或NMR确定。 DGCR8自身抑制的分子机制将使用体外方法检查。在特定的目标3中，将在人类细胞培养物中研究血红素-DGCR8相互作用的重要性和DGCR8在PRI-MIRNA加工中的自身抑制。医学启示：microRNA是一类新的基因，与重要的生物学和疾病过程有关。我们的研究旨在提高我们在人类基因组中找到这些基因的能力，以开发更好的治疗性microRNA，以调节疾病状态中的基因表达异常。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。