Molecular recognition and regulation in microRNA processing by the DGCR8 protein

描述(申请人提供)：microRNAs(MiRNAs)已被认为是一类重要的非编码RNA，参与各种生物和病理过程。广泛的长期目标是阐明长miRNA初级转录本(pri-miRNAs)被细胞因子特异性识别进行加工的分子机制，以及它们的成熟是如何通过涉及血红素的机制来调节的。具体地说，我们将关注一个关键因素的结构和功能--一种名为Dgcr8的人类RNA结合蛋白，它在DiGeorge综合征患者中杂合缺失。我们希望填补我们目前对miRNAs知识的主要空白：miRNA基因的一些关键功能尚未确定，因此无法从基因组序列准确预测miRNA基因；并且对miRNAs的加工是如何调控的知之甚少。特定目标1将阐明Dgcr8如何与pri-miRNAs相互作用。蛋白质-RNA界面将用生化和X射线结晶学方法进行检测。结合协作性的来源将用诱变方法进行研究。在特定目标2中，将揭示Dgcr8与血红素辅助因子之间的相互作用。将使用定点突变和筛选方法来鉴定对血红素结合重要的氨基酸残基。将使用X射线结晶学或核磁共振来确定血红素结合域的结构。我们将用体外方法研究Dgcr8自身抑制的分子机制。在特定目标3中，将研究血红素-Dgcr8相互作用和Dgcr8在pri-miRNA处理中的自身抑制在人类细胞培养中的重要性。医学启示：microRNAs是一类新的基因，参与重要的生物学和疾病过程。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，开发更好的治疗性microRNAs来调节疾病状态下的异常基因表达。我们的发现表明，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.