Regulation of cell cycle genes by K protein

描述（由申请人提供） 这是福格蒂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R 01 GM 45134-09。这项研究将主要在教授。 波兰华沙的耶日·奥斯特洛夫斯基实验室， FIRCA的提案 异质核核糖核蛋白K（hnRNP K）蛋白与 包括激酶、转录和翻译在内的多种分子 RNA和DNA。许多分子间的相互作用是由 细胞外信号，如生长因子。K蛋白已被证明 调节DNA转录和mRNA翻译的速率，通过直接 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， 信号转导途径到核酸指导过程的位点。 K蛋白结合编码细胞周期调节因子的mRNA，是 有丝分裂原应答激酶，并具有与细胞增殖相关的表达模式。 增殖基于这些观察，我们假设， 促有丝分裂信号K蛋白调节细胞周期基因的表达， DNA和RNA依赖性层。下面的目标将这个假设。 目标1。我们将鉴定K蛋白结合mRNA的所有组成部分， 细胞周期调节剂。与K蛋白免疫沉淀的mRNA， 血清处理的细胞将被用来产生一个复杂的探针， cDNA微阵列。将使用基于计算机的分析和凝胶迁移试验， 鉴定负责将K蛋白结合至靶标的RNA序列 成绩单 目标2。我们将确定细胞周期DNA位点的剧目，招募K 蛋白染色质免疫沉淀与抗K蛋白抗体和 微阵列的分析将用于识别招募K 蛋白质进入细胞周期。基于计算机的分析和凝胶移位 将使用检测来鉴定将K蛋白募集到细胞中的DNA序列。 靶位点。 目标3。我们将明确K蛋白在同源细胞调节中的作用， 循环DNA基因座和mRNA。蛋白质水平（蛋白质印迹法） (Aim#1-2）细胞周期调节因子及其mRNA（北方印迹）将被 在表达野生型和显性的 K蛋白阴性突变体。 鉴定表达依赖于K蛋白的细胞周期基因将 为未来的工作奠定基础，以定义精确的分子机制， 其中K蛋白调节和协调细胞周期基因DNA， mRNA指导的过程。拟议的研究将说明需要 增殖和肿瘤细胞改变K蛋白表达。

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.