Molecular recognition and regulation in microRNA processing by the DGCR8 protein

描述（由申请人提供）：MicroRNA（miRNAs）已被认为是一类重要的非编码RNA，参与多种生物学和病理学过程。广泛的，长期的目标是阐明的分子机制，通过该机制，长的miRNA初级转录本（pri-miRNAs）被细胞因子特异性识别加工，以及它们的成熟是如何通过涉及血红素的机制进行调节。具体来说，我们将集中在一个关键因素的结构和功能的人RNA结合蛋白DGCR 8是异源删除DiGeorge综合征患者。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识别，因此无法从基因组序列中准确预测miRNA基因;并且对miRNA的加工如何调控知之甚少。具体目标1将阐明DGCR 8如何与pri-miRNAs相互作用。将使用生物化学和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DGCR 8与血红素辅因子之间的相互作用。将使用定点诱变和筛选方法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来确定。将使用体外方法检查DGCR 8的自身抑制的分子机制。在具体目标3中，将在人细胞培养物中研究血红素-DGCR 8相互作用和DGCR 8在pri-miRNA加工中的自抑制的重要性。医学启示：MicroRNA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，开发更好的治疗microRNA，可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。

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.