Elements Controlling Fed-1 mRNA Stability and Light Regulated Translation

Oklahoma State University, Stillwater OK

Investigators

Abstract

0090666 Marie Petracek The 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.