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，因为这些因子的不受调控的产生会导致肿瘤发生和其他严重的临床症状。对于编码癌蛋白和细胞因子的mRNAs，这种调节包括由富含AU的元件(ARES)引导的细胞质mRNA的快速降解，ARES是一个多样化但进化保守的序列家族，编码于这些转录本的3‘非翻译区。我们的长期目标是确定ARES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，以及如何最终利用ARES的基因特异性特征作为治疗某些癌症和慢性炎症性疾病的新疗法的靶点。为了实现这些目标，将通过定量研究不同ARs与一些细胞质ARE结合蛋白之间相互作用的生化和细胞生物学后果，基于这样的假设，即每个ARE结合蛋白优先与ARE结合蛋白的子集相关，其中：(I)结合偏好由ARE内的初级和/或高阶RNA结构决定，以及(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，将通过三个具体目标来评估ARES模型和一组ARE结合蛋白之间相互作用的结构和功能后果。首先，将在体外鉴定重组反式因子与选定细胞mRNAs的ARES结合的RNA序列要求和结构后果，主要是通过将RNA突变与基于荧光的RNA-蛋白质平衡的定量分析相结合。其次，通过核酸酶足迹和荧光共振能量转移在体外鉴定不同细胞mRNAs中涉及ARs的高阶RNA结构，并定量评估它们在调节反式因子结合中的作用。最后，选定的反式作用因子调节报告mRNAs周转率的能力将在转基因细胞系统中通过异位过表达和/或RNA干扰介导的每个因子的耗尽来测试。总之，这些实验将定义ARES基因或基因家族的特定特征，这些特征决定了编码它们的mRNAs的细胞质命运(S)，并将确定哪个特定的反式作用因子(S)调节单个mRNAs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.