Mechanisms directing oncoprotein and cytokine mRNA decay

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，因为这些因子的失调产生有助于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（战神）指导的快速细胞质mRNA降解，富含AU的元件是在这些转录物的3'非翻译区内编码的序列的多样但进化上保守的家族。我们的长期目标是确定战神的大小和序列多样性如何在基因特异性水平上促进转录后调控，以及战神的基因特异性特征如何最终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战神与许多细胞质ARE结合蛋白之间相互作用的生物化学和细胞生物学后果来实现，基于每个ARE优先与ARE结合蛋白的子集缔合的假设，其中：（i）结合偏好由ARE内的初级和/或高级RNA结构决定，和（ii）ARE上的优先因子占据将mRNA导向许多可能的分解代谢或受保护的命运之一。为了验证这一假设，将通过三个特定目标评估模型战神与一组ARE结合蛋白之间相互作用的结构和功能后果。首先，RNA序列的要求和重组反式因子结合ARE从选定的细胞mRNA的结构后果将在体外鉴定，主要是通过耦合RNA诱变与定量，基于荧光的RNA-蛋白质平衡测定。第二，涉及战神从不同的细胞mRNA的高阶RNA结构将被确定在体外核酸酶足迹和荧光共振能量转移，和它们的作用，在调节反式因子结合定量评估。最后，通过异位过表达和/或RNA干扰介导的每种因子的耗竭，在转染的细胞系统中测试所选反式作用因子调节报告基因mRNA周转率的能力。总之，这些实验将定义战神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，并将鉴定哪些特定的反式作用因子介导个体mRNA的这些命运。

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.