RNA-binding protein CRD-BP in melanocyte biology

描述（由申请人提供）：本文提出的研究目的是了解RNA结合蛋白CRD-BP在黑素细胞生物学中的作用。小眼症相关转录因子（MITF）是黑素细胞发育的主要调节因子，也是恶性黑色素瘤生存和生长的关键因子。因此，了解控制MITF表达和功能的机制是非常重要的，因为它将确定黑素细胞发育和分化的关键组分。近年来，mRNA周转的调节作为控制基因表达的主要机制出现。mRNA的衰变速率响应于发育和环境信号，并由mRNA内的顺式作用元件和反式作用因子（如miRNA和RNA结合蛋白）决定。我们已经表明，MITF mRNA是CRD-BP的一种新的黑素细胞/黑色素瘤特异性靶点。我们证明了CRD-BP直接结合MITF mRNA的3 '-UTR，并阻止miR-340与其靶位点的结合，导致MITF转录物的稳定，从而诱导MITF表达和活性。CRD-BP是一种多功能RNA结合蛋白，可识别MITF、bTrCP 1、Gli 1、c-myc、IGF 2、tau和其他基因的mRNA。我们确定CRD-BP作为一个新的目标的b-连环蛋白/Tcf，和c-myc转录因子。我们的初步数据表明，在响应β-连环蛋白信号，CRD-BP结合和稳定的c-myc和bTrCP 1的mRNA。CRD-BP是必不可少的诱导c-myc和bTrCP 1的b-连环蛋白信号。CRD-BP的转录上调b-连环蛋白/Tcf因此可能有助于多效性效应的Wnt信号通路，并可能提供的假设，CRD-BP可能有一个重要的功能Wnt/b-连环蛋白依赖的黑素细胞的发展的基础。除了Wnt信号的调节外，我们的初步结果表明，缺氧也诱导CRD-BP的表达。考虑到皮肤的生理缺氧环境，这种新的CRD-BP表达和功能调节模式可能在黑素细胞生长和发育中发挥重要作用。我们推测，通过影响MITF和其他mRNA的稳定性，CRD-BP对正常黑素细胞发育至关重要。我们拟研究CRD-BP在黑素细胞生物学中的作用及其调控机制和意义。根据这些目标，具体目标是：1.目的：明确黑素细胞生长和分化对CRD-BP的需求。2.探讨低氧对CRD-BP的调节机制。3.分析缺氧对黑素细胞CRD-BP调节的病理生理意义。

DESCRIPTION (provided by applicant): The goal of studies proposed here is to understand the role of the role of RNA-binding protein, CRD-BP, in melanocyte biology. Microphtalmia-associated transcription factor (MITF) is a principal regulator of melanocyte development and a pivotal factor in the survival and growth of malignant melanomas. Therefore understanding the mechanisms controlling the expression and function of MITF is extremely important as it will identify key components for melanocyte development, and differentiation. Regulation of mRNA turnover emerged in recent years as a major mechanism controlling gene expression. mRNA decay rates are responsive to developmental and environmental signals and dictated by cis-acting elements within the mRNA and by trans-acting factors, such as miRNAs and RNA-binding proteins. We have shown that MITF mRNA is a novel melanocyte/melanoma-specific target of CRD-BP. We demonstrated that CRD-BP directly binds to the 3'-UTR of MITF mRNA and prevents the binding of miR-340 to its target sites resulting in stabilization of the MITF transcript that leads to induction of MITF expression and activities. CRD-BP is a multifunctional RNA-binding protein that recognizes the mRNAs of MITF, bTrCP1, Gli1, c-myc, IGF2, tau, and other genes. We identified CRD-BP as a novel target of b-catenin/Tcf, and c-myc transcription factors. Our preliminary data demonstrate that in response to b-catenin signaling, CRD-BP binds and stabilizes mRNAs of c-myc and bTrCP1. CRD-BP is essential for induction of c-myc and bTrCP1 by b-catenin signaling. Transcriptional upregulation of CRD-BP by b-catenin/Tcf may therefore contribute to pleiotropic effects of the Wnt signaling pathway and could provide the foundation for the hypothesis that CRD-BP might have a significant function in Wnt/b-catenin-dependent melanocyte development. In addition to the regulation by Wnt signaling, our preliminary results show that hypoxia also induces the expression of CRD-BP. Taking into consideration the physiologically hypoxic environment of the skin, this novel mode of regulation of expression and function of CRD-BP could play a significant role in melanocyte growth and development. We hypothesize that by influencing the stability of MITF and other mRNAs, CRD-BP is essential for normal melanocyte development. We propose to study the role of CRD-BP in melanocyte biology, and the mechanisms and significance of its regulation in melanocytes. Pursuant to these goals, the specific aims are: 1. To define the requirement for CRD-BP in the growth and differentiation of melanocytes. 2. To delineate the mechanism(s) of CRD-BP regulation by hypoxia. 3. To analyze the (patho)physiologic significance of CRD-BP regulation by hypoxia in melanocytes.