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.

C-myc基因表达调控是细胞增殖的重要决定因素 增殖和分化并避免肿瘤细胞行为。 调节通常发生在mRNA水平，取决于标记的 mRNA的不稳定性，其快速性，并经常 转录后 初步研究表明，C2小鼠成肌细胞 表达丰富的c-myc mRNA，这种mRNA的周转是迅速的， 依赖于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水平的下降， 这是由于加速周转，这也取决于翻译， C末端蛋白质编码序列。 本提案的目的是 了解这些转录后机制调节c-myc 表情(I)翻译的结构决定因素的描述- 依赖性c-myc mRNA不稳定性将通过检测myc 删除和融合mRNA的诱导放线菌酮，这已经被 显示是由于药物抑制翻译使mRNA稳定， 说明myc基因的mRNA不稳定 点与移帧研究 突变体将指示核苷酸序列或编码的氨基酸 该元素的序列是不稳定性决定因素。 类似的 方法将用于描绘靶向c-myc的结构元件 mRNA在C2分化过程中的调节。 这个任务可以简化 通过仅从放线菌酮中选择信息量最大的myc基因， 在繁琐的C2分化试验中进行测试的研究，因为 初步研究表明，监管的决定因素和 不稳定性可能是相同的。(II)这些c-myc mRNA的鉴定 结构决定因素之后，将描述其 意义 目前的混乱造成的多样性， 到目前为止，在c-myc mRNA中鉴定出的假定的不稳定元件将是 通过确定哪个候选元素实际上影响 稳态水平的c-myc mRNA通过指定其周转率。 的 c-myc mRNA不稳定性的预防依赖性机制的患病率 在分化过程中的调节将通过检查它们的 在其他类型的细胞中使用。 如果c-myc以外的mRNAs受 在分化过程中，它们可能具有相同的机制， 调节基序，并在此基础上使用适当的搜索进行识别 或克隆策略。(III)关于顺式作用基序的知识 调节c-myc mRNA水平将被用来表征 涉及的因素。 一种方法是建立一个体外系统， c-myc mRNA降解忠实地再现了体内事件， 用于表征和纯化这些因素。 如果一个核苷酸序列 形成调控基序，识别结合这种基序的因子， 序列提供了另一种方法。 这些研究有望揭示 参与细胞mRNA周转的调节，并揭示了 转录后c-myc mRNA调控的分子基础， 细胞对生长和分化的正常反应的要素 刺激。