Elements Controlling Fed-1 mRNA Stability and Light Regulated Translation

控制 Fed-1 mRNA 稳定性和光调节翻译的元素

基本信息

  • 批准号:
    0090666
  • 负责人:
  • 金额:
    $ 40万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
    Standard Grant
  • 财政年份:
    2001
  • 资助国家:
    美国
  • 起止时间:
    2001-03-01 至 2006-02-28
  • 项目状态:
    已结题

项目摘要

0090666 Marie PetracekThe ability to recognize and respond to extracellular cues is essential for all living organisms. In plants, one of the most important cues is light, which regulates growth and development through transcriptional and post-transcriptional regulation of nuclear and chloroplast gene expression. One of the best examples of post-transcriptional regulation of a nuclear-encoded mRNA is that of the pea Fed-1 gene, which encodes the chloroplast ferredoxin protein. Fed-1 is regulated by light at the levels of mRNA stability and translation. A decrease in photosynthesis results in a rapid decrease in the cytoplasmic translation of Fed-1 mRNA followed by degradation of the Fed-1 mRNA. Importantly, the polyribosome association of a subset of other nuclear-encoded mRNAs also declines rapidly in the absence of photosynthesis, suggesting that photosynthetic regulation of cytoplasmic translation, and perhaps subsequent mRNA decay, is an important mechanism for light regulation of nuclear gene expression. Further analysis of the light-responsive properties of Fed-1 mRNA will yield important information on how the translational apparatus responds to changes in photosynthesis, on how specificity of these translational changes is conferred on a subset of mRNAs, and on how translation and mRNA degradation are coupled. For Fed-1, light-regulated mRNA stability and translation appear to be conferred by two separate elements. The first element, necessary for destabilization of the mRNA in the dark, is within a region of the Fed-1 5' UTR that contains a (CATT)4 repeat. The (CATT)4 repeat and the surrounding region will be mutated to delineate the mRNA instability element, to test if the function of the instability element is position dependent, and to determine if the element is sufficient to destabilize non-light-regulated mRNAs. To identify proteins involved in regulated Fed-1 mRNA degradation, the instability element will be used as a bait in a yeast 3-hybrid screen. The second element in Fed-1 mRNA, required for light-regulated translation, may be in the Fed-1 mRNA coding sequence. The translational control element will be further delimited by mutagenesis and, once characterized, the element will be added to non-light regulated mRNAs to determine the minimal sequence sufficient to repress translation in response to darkness. It is possible that the Fed-1 "translational control" element is an mRNA localization element rather than an element that directly controls translation. To determine if the translational control element is an mRNA localization sequence, Fed-1 mRNAs that are wild-type or mutant for translational regulation by light will be localized using a GFP system adapted to plants. These studies will extend our rudimentary knowledge of post-transcriptional gene regulation in plants and provide a foundation to uncover the mechanism of photosynthetic control of cytoplasmic translation in plants.
0090666玛丽·彼得雷克斯识别和响应细胞外信号的能力对所有活着的有机体来说都是必不可少的。在植物中,光是最重要的信号之一,它通过转录和转录后调控核和叶绿体基因的表达来调节生长和发育。对核编码的mRNA进行转录后调控的最好例子之一是豌豆FED-1基因,它编码叶绿体铁氧还蛋白。FED-1在mRNA稳定性和翻译水平上受光的调控。光合作用的下降导致FED-1mRNA在细胞质中的翻译迅速减少,随后FED-1mRNA的降解。重要的是,在没有光合作用的情况下,其他核编码的mRNAs的多聚核糖体结合也会迅速下降,这表明光合作用对细胞质翻译的调节,以及随后的mRNA衰变,是光调节核基因表达的重要机制。进一步分析FED-1mRNA的光响应特性将产生关于翻译机构如何响应光合作用变化的重要信息,这些翻译变化的特异性如何被赋予mRNAs的子集,以及翻译和mRNA降解是如何耦合的。对于FED-1,光调节的mRNA稳定性和翻译似乎由两个独立的元件决定。第一个元件是在黑暗中使mRNA不稳定所必需的,它位于FED-1 5‘UTR的一个包含(Catt)4重复的区域内。(Catt)4重复序列及其周围区域将发生突变,以描绘出mRNA不稳定元件,测试不稳定元件的功能是否具有位置依赖性,并确定该元件是否足以破坏非光调控的mRNAs的稳定性。为了确定参与FED-1mRNA降解的蛋白质,不稳定元件将被用作酵母3-杂交筛选的诱饵。FED-1mRNA中的第二个元件可能在FED-1mRNA编码序列中,这是光调控翻译所必需的。翻译控制元件将通过突变进一步界定,一旦表征,该元件将被添加到非光调节的mRNAs中,以确定足以抑制黑暗反应的翻译的最小序列。FED-1“翻译调控”元件可能是一种mRNA本地化元件,而不是直接控制翻译的元件。为了确定翻译控制元件是否为mRNA定位序列,将使用适合植物的GFP系统对野生型或突变型FED-1mRNAs进行定位。这些研究将扩展我们对植物转录后基因调控的基础知识,并为揭示植物光合作用控制细胞质翻译的机制提供基础。

项目成果

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