POST-TRANSCRIPTIONAL REGULATION OF C-MYC MRNA

调控的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。 调控通常发生在信使核糖核酸水平，取决于标记的 信使核糖核酸的不稳定，因为它的快速和频繁的 转录后。初步研究表明，C2小鼠成肌细胞 表达丰富的c-myc信使核糖核酸，该信使核糖核酸周转迅速。 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， 这是由于加速的营业额，这也取决于翻译 C-末端蛋白编码序列。这项提议的目标是 了解这些转录后调控c-myc的机制 表情。(I)界定翻译的结构决定因素-- 依赖的c-myc基因的不稳定性将通过检测myc来实现 环己亚胺诱导的缺失和融合mRNAs，已被 被证明是由于药物抑制翻译和转录而使mRNA稳定 表明存在不稳定的myc基因。点移法和移标法的研究 突变体将表明核苷酸序列或编码的氨基酸 该元素的序列是不稳定的决定因素。一种类似的 将使用一种方法来描述以c-myc为靶点的结构元件 在C2分化过程中进行调控的基因。这项任务可以简化。 通过仅从放线菌亚胺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，监管和监管的决定因素 不稳定可能也是一样的。(Ii)这些c-myc基因的鉴定 结构决定因素之后将描述它们的特征 意义。目前的混乱是由多样性造成的 到目前为止在c-myc mRNA中发现的可能的不稳定元件将是 通过确定哪个候选元素实际影响 通过指定其周转率来确定c-myc mRNA的稳态水平。这个 C-myc信使核糖核酸不稳定的翻译依赖机制 而差异化过程中的监管将通过检查其 在其他单元格类型中的使用。如果c-myc以外的mRNAs受 在分化过程中，它们的机制相同，可能共享myc mrna。 并在此基础上使用适当的搜索进行识别 或者克隆策略。(3)获得关于顺位代理主题的知识 调节c-myc mRNA水平将被用来表征交易 涉及的因素。一种方法是建立一个体外系统 C-myc mRNA的降解如实地概括了体内事件，并可以 用来表征和提纯这些因素。如果一个核苷酸序列 形成了调控的主题，确定了约束这一主题的因素 Sequence提供了另一种方法。这些研究有望揭示各种因素 参与调节细胞信使核糖核酸的周转，并披露 转录后c-myc基因调控的分子基础 细胞对生长和分化的正常反应的要素 刺激物。

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.