Mechanisms directing oncoprotein and cytokine mRNA decay

指导癌蛋白和细胞因子 mRNA 衰减的机制

• 批准号: 6895416
• 负责人: Gerald M. Wilson
• 金额: $ 26.43万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6895416
• 关键词: RNase protection assay; binding proteins; cell line; cytokine; fluorescence resonance energy transfer; messenger RNA; nucleic acid metabolism; nucleic acid quantitation /detection; nucleic acid sequence; nucleic acid structure; oncoproteins; posttranscriptional RNA processing; protein binding; transcription factor; transfection

DESCRIPTION (provided by applicant): In mammals, the expression of proteins regulating cell proliferation and differentiation is tightly controlled, since disregulated production of these factors contributes to oncogenesis and other serious clinical syndromes. For mRNAs encoding oncoproteins and cytokines, this regulation includes rapid cytoplasmic mRNA degradation directed by AU-rich elements (AREs), a diverse but evolutionarily conserved family of sequences encoded within the 3' untranslated regions of these transcripts. Our long-term objectives are to determine how the size and sequence diversity of AREs contributes to post-transcriptional regulation at the gene-specific level, and how gene-specific characteristics of AREs might ultimately be exploited as targets for novel therapies to treat some cancers and chronic inflammatory diseases. These goals will be pursued by quantitatively examining the biochemical and cell biological consequences of interactions between different AREs and a number of cytoplasmic ARE-binding proteins, based on the hypothesis that each ARE preferentially associates with a subset of ARE-binding proteins where: (i) binding preference is dictated by primary and/or higher-order RNA structures within the ARE, and (ii) preferential factor occupancy on the ARE directs the mRNA to one of a number of possible catabolic or protected fates. To test this hypothesis, the structural and functional consequences of interactions between model AREs and a panel of ARE-binding proteins will be assessed through three Specific Aims. First, the RNA sequence requirements and structural consequences of recombinant trans-factor binding to AREs from selected cellular mRNAs will be identified in vitro, largely by coupling RNA mutagenesis with quantitative, fluorescence-based assays of RNA-protein equilibria. Second, higher-order RNA structures involving AREs from different cellular mRNAs will be identified in vitro by nuclease footprinting and fluorescence resonance energy transfer, and their role in modulating trans-factor binding quantitatively assessed. Finally, the ability of selected trans-acting factors to modulate the turnover rates of reporter mRNAs will be tested in a transfected cell system by ectopic overexpression and/or RNA interference-mediated depletion of each factor. Together, these experiments will define gene- or gene family-specific features of AREs that dictate the cytoplasmic fate(s) of mRNAs encoding them, and will identify which specific trans-acting factor(s) mediate these fates for individual mRNAs.

描述（由申请人提供）： 在哺乳动物中，调节细胞增殖和分化的蛋白质的表达受到严格控制，因为这些因子的产生失调会导致肿瘤发生和其他严重的临床综合征。对于编码癌蛋白和细胞因子的 mRNA，这种调节包括由富含 AU 的元件 (ARE) 指导的快速细胞质 mRNA 降解，ARE 是这些转录物 3' 非翻译区域内编码的多样化但进化保守的序列家族。我们的长期目标是确定 ARE 的大小和序列多样性如何促进基因特异性水平的转录后调控，以及 ARE 的基因特异性特征最终如何被利用作为治疗某些癌症和慢性炎症性疾病的新疗法的靶点。这些目标将通过定量检查不同 ARE 和许多细胞质 ARE 结合蛋白之间相互作用的生化和细胞生物学后果来实现，基于每个 ARE 优先与 ARE 结合蛋白子集关联的假设，其中：(i) 结合偏好由 ARE 内的初级和/或更高阶 RNA 结构决定，以及 (ii) ARE 上的优先因子占据指示 mRNA 与许多可能的分解代谢或受保护的命运之一有关。为了检验这一假设，模型 ARE 与一组 ARE 结合蛋白之间相互作用的结构和功能后果将通过三个具体目标进行评估。首先，重组反式因子与选定细胞 mRNA 中的 ARE 结合的 RNA 序列要求和结构后果将在体外鉴定，主要是通过将 RNA 诱变与基于荧光的 RNA-蛋白质平衡定量测定相结合。其次，涉及来自不同细胞 mRNA 的 ARE 的高阶 RNA 结构将通过核酸酶足迹和荧光共振能量转移在体外进行鉴定，并定量评估它们在调节反式因子结合中的作用。最后，将在转染的细胞系统中通过异位过度表达和/或RNA干扰介导的各因子的消耗来测试所选反式作用因子调节报告基因mRNA更新率的能力。总之，这些实验将定义 ARE 的基因或基因家族特异性特征，这些特征决定编码它们的 mRNA 的细胞质命运，并将确定哪些特定的反式作用因子介导单个 mRNA 的这些命运。