Regulation of cell cycle genes by K protein

描述(由申请人提供) 这是Fogarty国际研究中心的拨款申请 合作奖(FIRCA)，以补充目前资助的NIH项目，赠款 编号R01GM 45134-09。这项拟议的研究将主要在科罗拉多教授进行。 Jerzy Ostrowski在波兰华沙的实验室， 这份FIRCA提案。 异质核糖核蛋白K，hnRNP K蛋白与 包括激酶、转录和翻译在内的多种分子 因子、RNA和DNA。这些分子相互作用中的许多是由 细胞外信号，如生长因子。K蛋白已被证明是 直接调节DNA转录和信使核糖核酸的翻译速率 分别与富含CT和Cu的核苷酸序列相互作用。它有 有人认为，K蛋白作为一个对接平台，起到了连接 去往核酸调控过程位点的信号转导途径。 K蛋白结合编码细胞周期调节因子的mRNAs，是一种底物 丝裂原反应蛋白，并具有与细胞相关的表达模式 扩散。基于这些观察，我们假设为了回应 丝裂原信号K蛋白调节细胞周期基因的表达(S) 依赖DNA和RNA的层级。以下目标将验证这一假设。 目的#1.我们将确定编码K蛋白结合mRNAs的谱系 细胞周期调节器。静止期K蛋白免疫沉淀的mRNAs 经过血清处理的细胞将被用来产生一个复杂的探针来分析 CDNA微阵列。将使用基于计算机的分析和凝胶位移分析来 确定负责将K蛋白与靶结合的RNA序列 成绩单。 目的#2.我们将确定细胞周期DNA基因座中招募K 蛋白。抗K蛋白抗体与染色质免疫沉淀 微阵列的图谱将被用来识别招募K 细胞中的蛋白质进入细胞周期。基于计算机的分析和凝胶置换 检测将被用来鉴定将K蛋白招募到 目标轨迹。 目的#3.我们将确定K蛋白在同源细胞调节中的作用 循环DNA基因座和mRNAs。所选蛋白的蛋白质水平(蛋白质印迹) (目标#1-2)细胞周期调节因子及其mRNAs(Northern印迹) 在血清刺激细胞中评估表达野生型和显性 K蛋白阴性突变体。 K蛋白依赖意志表达的细胞周期基因的鉴定 为未来的工作奠定基础，通过以下方式定义精确的分子机制 哪个K蛋白调节和协调细胞周期基因DNA-和 信使核糖核酸调控的过程。拟议的研究将解释是否需要 增殖和肿瘤细胞改变K蛋白的表达。

DESCRIPTION (provided by applicant) This is a grant application for the Fogarty International Research Collaboration Award (FIRCA) to supplement presently funded NIH project, grant number R01GM 45134-09. The proposed research will be done primarily in Prof. Jerzy Ostrowski's Laboratory in Warsaw, Poland, the foreign collaborator of this FIRCA proposal. The heterogeneous nuclear ribonucleoprotein K, hnRNP K, protein interacts with a diversity of molecules including kinases, transcription and translation factors, RNA and DNA. Many of these molecular interactions are regulated by extracellular signals, such as growth factors. K protein has been shown to regulate rates of DNA transcription and mRNA translation through direct interaction with CT- and CU-rich nucleotide sequences, respectively. It has been suggested that K protein, acting as a docking platform, serves to link signal transduction pathways to sites of nucleic acid-directed processes. K protein binds mRNAs that encode cell cycle regulators, is a substrate for mitogen-responsive kinases, and has an expression pattern correlated with cell proliferation. Based on these observations we postulate that in response to mitogenic signals K protein regulates expression of cell cycle gene(s) at both DNA- and RNA-dependent tiers. The following aims will this hypothesis. Aim#1. We will identify the repertoire of K protein-binding mRNAs that encode cell cycle regulators. mRNAs immunoprecipitated with K protein from quiescent and serum-treated cells will be used to generate a complex probe to profile cDNA microarrays. Computer-based analysis and gel-shift assay will be used to identify RNA sequences that are responsible for binding K protein to the target transcripts. Aim#2. We will identify the repertoire of cell cycle DNA loci that recruit K protein. Chromatin immunoprecipitation with anti-K protein antibody and profiling of microarrays will be used to identify DNA loci that recruit K protein in cells entering cell cycle. Computer-based analysis and gel-shift assays will be used to identify DNA sequences that recruit K protein to the target loci. Aim#3. We will define the role of K protein in the regulation of cognate cell cycle DNA loci and mRNAs. Protein levels (Western blots) of the selected (Aim#1-2) cell cycle regulators and their mRNAs (Northern blots) will be assessed in serum stimulated cells that express wild type and a dominant negative K protein mutant. Identification of cell cycle genes whose expression is K protein-dependent will set the stage for future work to define the precise molecular mechanisms by which K protein regulates and coordinates cell cycle gene DNA- and mRNA-directed processes. The proposed studies will explain the need of proliferating and tumor cells to alter K protein expression.