Analysis of the Ribonucleases of Arabidopsis thaliana

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0096394 Pam GreenGene expression is a multi-step process fundamental to all phases of plant growth and development. The first step in the process, transcription, has thus far received the most attention and therefore is the most well understood at the molecular level. However, it is clear that post-transcriptional events can have profound effects on gene expression. One prominent post-transcriptional event is that of mRNA degradation. In plant and other eukaryotic cells, the number of genes considered to be regulated at the level of mRNA stability is increasing rapidly. Despite the importance of mRNA stability in gene expression, the enzymes that catalyze mRNA decay in cells of plants and other multicellular eukaryotes have yet to be identified. This project will address the function of several potential mRNA degrading enzymes (mRNases) in Arabidopsis using functional genomic and biochemical analysis. The hypothesis is that these enzymes catalyze different steps in mRNA decay: AtPARN removes the poly(A) tail, AtDCP1 with co-factor AtDCP2 removes the cap, and AtXRN4 degrades the remainder of the RNA starting from one end. Although the DCPs and XRN1 facilitate the latter two steps of most cytoplasmic mRNAs in yeast, their functions in multicellular eukaryotes such as mammals and plants remain an open question because plants and mammals exhibit differences in mRNA decay compared to that in yeast. Nevertheless, the presence of homologous genes in plants and animals, as well as other observations, suggests that yeast will provide some clues about components common to all eukaryotes. Therefore, what is needed is a careful analysis of the general mRNA decay machinery in a multicellular eukaryote to elucidate the fundamentals of how mRNA stability is controlled in complex systems versus yeast. Arabidopsis has been chosen as the model system because of the experimental tools available and the importance of mRNA stability in establishing plant gene expression levels. To further understanding of the mRNA decay roles of AtPARN, AtDCP1, and AtXRN4, and their potential substrates genome-wide, this project will 1) characterize the enzymes biochemically and examine their intracellular locations, 2) obtain knockout or "conditional" knockout mutants in the corresponding genes, and 3) analyze the most informative of these mutants via DNA microarray analysis and other RNA analyses to identify mRNA substrates. The roles and potential targets of these putative mRNases have never been studied on a global scale nor have the corresponding genes been inactivated in any multicellular eukaryote. Therefore, these experiments should have broad significance. Moreover, for some of these enzymes, characteristics unique to plants have been discovered; thus this work has the potential to pinpoint aspects of mRNA decay that are novel and plant specific.