Molecular recognition and regulation in microRNA processing by the DGCR8 protein

描述（由申请人提供）：MicroRNAs （miRNAs）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。广泛而长期的目标是阐明长miRNA初级转录物（pri-miRNA）被细胞因子特异性识别并加工的分子机制，以及它们的成熟如何通过涉及血红素的机制进行调节。具体来说，我们将关注一个关键因子的结构和功能——一种被称为DGCR8的人类RNA结合蛋白，它在DiGeorge综合征患者中被杂合删除。我们希望填补我们目前对miRNA知识的主要空白：miRNA基因的一些关键特征尚未被鉴定，因此无法从基因组序列中准确预测miRNA基因；而对于mirna的加工是如何被调控的，我们知之甚少。特异性目的1将阐明DGCR8如何与pri- mirna相互作用。蛋白质- rna界面将使用生化和x射线晶体学方法进行检查。结合协同性的起源将利用诱变方法进行研究。在具体目标2中，DGCR8和血红素辅助因子之间的相互作用将被揭示。对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进行鉴定。血红素结合区域的结构将使用x射线晶体学或核磁共振来确定。DGCR8自身抑制的分子机制将采用体外方法进行研究。在特定目标3中，将在人类细胞培养中研究血红素-DGCR8相互作用和DGCR8在pri-miRNA加工中的自我抑制的重要性。医学意义：MicroRNAs是一类新的基因，参与重要的生物和疾病过程。我们的研究旨在提高我们在人类基因组中发现这些基因的能力，以开发更好的治疗性microrna，可以调节疾病状态下异常基因的表达。我们发现血红素可能参与调节microrna的加工过程，这表明它们可以用作microrna相关疾病的治疗剂。

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.