POSTTRANSCRIPTIONAL REGULATION OF C-MYC MRNA

C-MYC mRNA 的转录后调控

• 批准号: 2184557
• 负责人: William M Lee
• 金额: $ 20.57万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2184557
• 关键词: cell differentiation; cell free system; cell type; cycloheximide; frameshift mutation; gene expression; genetic regulatory element; laboratory mouse; messenger RNA; myoblasts; neoplastic transformation; nucleic acid sequence; oncogenes; point mutation; posttranscriptional RNA processing; protein degradation; transfection

Regulated C-myc gene expression is an important determinant of cell proliferation and differentiation and averts neoplastic cell behavior. Regulation usually occurs at the mRNA level, depends on the marked instability of the mRNA for its rapidity and is frequently post-transcriptional. Preliminary studies show that C2 murine myoblasts express abundant c-myc mRNA and that the turnover of this mRNA is rapid and depends on translation of C-terminal protein-coding sequences. Differentiation of C2 cells is accompanied by a fall in c-myc mRNA levels, which is due to accelerated turnover that also depends on translation of C-terminal protein-coding sequences. The goal of this proposal is to understand these post-transcriptional mechanisms regulating c-myc expression. (I) Delineation of the structural determinant of translation- dependent c-myc mRNA instability will be accomplished by testing myc deletion and fusion mRNAs for induction by cycloheximide, which has been shown to be due to mRNA stabilization by drug inhibition of translation and is indicative of an unstable myc mRNA. Study of point and frame-shift mutants will indicate if the nucleotide sequence or the encoded amino acid sequence of this element is the instability determinant. A similar approach will be used to delineate the structural element targeting c-myc mRNA for regulation during C2 differentiation. This task may be simplified by selecting only the most informative myc genes from the cycloheximide studies for testing in the cumbersome C2 differentiation assay, since preliminary studies indicate that the determinants of regulation and instability may be the same. (II) Identification of these c-myc mRNA structural determinants will be followed by characterization of their significance. The current confusion resulting from the multiplicity of putative destabilizing elements identified in c-myc mRNA to date will be resolved by determining which candidate element actually influences the steady-state level of c-myc mRNA by specifying its rate of turnover. The prevalence of translation-dependent mechanisms for c-myc mRNA instability and regulation during differentiation will be determined by examining their usage in other cell types. If mRNAs other than c-myc are regulated by the same mechanism during differentiation, they may share the myc mRNA regulatory motif and be identifiable on this basis using appropriate search or cloning strategies. (III) Knowledge gained about the cis-acting motifs regulating c-myc mRNA levels will be used to characterize the transacting factors involved. One approach will be to establish an in vitro system of c-myc mRNA degradation that faithfully recapitulates in vivo events and can be used to characterize and purify these factors. If a nucleotide sequence forms the regulatory motif, identification of factors that bind this sequence offers another approach. These studies promise to reveal factors involved in the regulation of cellular mRNA turnover and to disclose the molecular basis of post-transcriptional c-myc mRNA regulation, a key element of the cell's normal response to growth and differentiation stimuli.

调节的C-MYC基因表达是细胞的重要决定因素 增殖和分化并避免肿瘤细胞行为。 调节通常在mRNA水平上发生，取决于标记的 mRNA的速度不稳定，并且经常是 转录后。 初步研究表明C2鼠肌细胞 表达大量的C-Myc mRNA，并且该mRNA的营业额很快，并且 取决于C末端蛋白质编码序列的翻译。 C2细胞的分化伴随着C-MYC mRNA水平下降， 这是由于加速营业额，也取决于翻译 C末端蛋白质编码序列。 该提议的目的是 了解这些调节C-MYC的转录后机制 表达。 （i）划定翻译的结构决定因素 - 依赖的C-MYC mRNA不稳定性将通过测试MYC实现 删除和融合mRNA通过环己酰亚胺诱导，这是 证明是由于药物抑制翻译和 表示不稳定的MYC mRNA。 点和框架迁移的研究 突变体将指示核苷酸序列或编码的氨基酸是 该元素的序列是不稳定性的决定因素。 类似 方法将用于描述针对C-MYC的结构元件 在C2分化过程中调节的mRNA。 此任务可以简化 通过仅从环己酰胺中选择最有用的MYC基因 在繁琐的C2分化测定中进行测试的研究，因为 初步研究表明，调节和 不稳定可能是相同的。 （ii）鉴定这些C-Myc mRNA 结构决定因素将在其表征之后进行 意义。 当前的混乱是由 到目前 通过确定哪些候选元素实际影响 C-MYC mRNA的稳态水平通过指定其营业率。 这 C-MYC mRNA不稳定性的翻译依赖性机制的流行率 在差异化期间的调节将通过检查其 在其他细胞类型中使用。 如果C-MYC以外的mRNA受到 分化过程中相同的机制，它们可能共享MYC mRNA 监管主题，并在此基础上使用适当的搜索来识别 或克隆策略。 （iii）获得有关顺式作用基序的知识 调节C-MYC mRNA水平将用于表征交易 涉及的因素。 一种方法是建立一个体外系统 c-myc mRNA降解，忠实地概括体内事件，可以 用于表征和净化这些因素。 如果核苷酸序列 形成调节基序，识别与此结合的因素 序列提供了另一种方法。 这些研究有望揭示因素 参与细胞mRNA转移的调节，并披露 转录后C-MYC mRNA调节的分子基础，这是一个钥匙 细胞对生长和分化的正常响应的元素 刺激。