RNA-binding protein CRD-BP in melanocyte biology

描述（由申请人提供）：这里提出的研究目标是了解rna结合蛋白CRD-BP在黑素细胞生物学中的作用。microphtalmia相关转录因子（MITF）是黑素细胞发育的主要调节因子，是恶性黑素瘤存活和生长的关键因子。因此，了解MITF的表达和功能控制机制是非常重要的，因为它将确定黑素细胞发育和分化的关键成分。近年来，mRNA周转调控成为调控基因表达的主要机制。mRNA的衰变速率受发育和环境信号的影响，并受mRNA内的顺式作用元件和反式作用因子（如mirna和rna结合蛋白）的支配。我们已经证明，MITF mRNA是一种新的黑素细胞/黑素瘤特异性靶向CRD-BP。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，从而导致MITF转录物的稳定，从而诱导MITF的表达和活性。CRD-BP是一种多功能rna结合蛋白，可识别MITF、bTrCP1、Gli1、c-myc、IGF2、tau等基因的mrna。我们发现CRD-BP是b-catenin/Tcf和c-myc转录因子的新靶点。我们的初步数据表明，在响应b-catenin信号传导时，CRD-BP结合并稳定c-myc和bTrCP1的mrna。CRD-BP是通过b-catenin信号传导诱导c-myc和bTrCP1的必要条件。因此，b-catenin/Tcf对CRD-BP的转录上调可能有助于Wnt信号通路的多向性作用，并可能为CRD-BP可能在Wnt/b-catenin依赖性黑素细胞发育中发挥重要作用的假设提供基础。除了Wnt信号的调控外，我们的初步结果表明，缺氧也诱导了CRD-BP的表达。考虑到皮肤的生理性缺氧环境，这种新的调节CRD-BP表达和功能的模式可能在黑素细胞的生长和发育中发挥重要作用。我们假设，通过影响MITF和其他mrna的稳定性，CRD-BP对正常的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，及其在黑素细胞中的调控机制和意义。根据这些目标，具体目标是：1。定义黑素细胞生长分化过程中对CRD-BP的需求。2. 探讨缺氧对crp - bp调节的机制。3. 探讨缺氧对黑素细胞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.