Elements Controlling Fed-1 mRNA Stability and Light Regulated Translation

识别和响应细胞外信号的能力对所有生物体来说都是必不可少的。在植物中，最重要的线索之一是光，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。核编码mRNA转录后调控的最好例子之一是豌豆的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调控。光合作用的减少导致Fed-1 mRNA的胞质翻译迅速减少，随后Fed-1 mRNA降解。重要的是，在没有光合作用的情况下，其他核编码mRNA子集的多核糖体关联也会迅速下降，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。对Fed-1 mRNA光响应特性的进一步分析将提供重要信息，了解翻译装置如何响应光合作用的变化，这些翻译变化的特异性如何赋予mRNA子集，以及翻译和mRNA降解如何耦合。对于Fed-1，光调控的mRNA稳定性和翻译似乎是由两个独立的元件赋予的。第一个元件是在黑暗中使mRNA不稳定所必需的，它位于fed - 15 ' UTR中包含（CATT）4重复序列的区域内。（CATT）4重复和周围区域将发生突变，以描绘mRNA不稳定元件，以测试不稳定元件的功能是否依赖于位置，并确定该元件是否足以使非光调节mRNA不稳定。为了鉴定参与调控的Fed-1 mRNA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-1 mRNA中光调控翻译所需的第二个元件可能位于Fed-1 mRNA编码序列中。翻译控制元件将通过诱变进一步划分，一旦确定特征，该元件将被添加到非光调节的mrna中，以确定在黑暗条件下足以抑制翻译的最小序列。Fed-1“翻译控制”元件可能是mRNA定位元件，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系统定位用于光翻译调节的野生型或突变型Fed-1 mRNA。这些研究将扩展我们对植物转录后基因调控的基本认识，并为揭示植物胞质翻译的光合控制机制提供基础。

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.