Analysis of the Ribonucleases of Arabidopsis thaliana

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The regulation of genes is of fundamental importance for all phases of growth and development. Genes function by producing mRNA molecules that carry genetic information to the machinery that makes proteins, which are the products of genes. Because the amount of mRNA is generally related to the amount of protein produced, many gene regulatory mechanisms control the synthesis or degradation rates of mRNAs. This project focuses on two types of ribonucleases (RNases) that degrade mRNA in the model plant Arabidopsis. Work on one type, the 5'-3' exoribonucleases, will concentrate primarily on the function of AtXRN4. Previous work funded by NSF showed that AtXRN4 preferentially degrades specific mRNA molecules, including a subset of the mRNAs targeted for silencing by very small RNAs called miRNAs. To better understand the basis of this specificity, this project will address the importance of mRNA location within the cell and of sequences within mRNAs. Deadenylases are the second type of RNase to be studied. Both Arabidopsis Poly(A) Ribonuclease (AtPARN) and the Arabidopsis family of Ccr4p deadenylases will be examined to determine which mRNAs they preferentially degrade. This will be accomplished by using mutant plants lacking one or more of these deadenylases for biochemical and molecular biological experiments such as DNA microarray analysis, a technology for determining mRNA levels for thousands of genes at once. This work will have broad impact beyond the plant field because similar questions have yet to be answered in other multicellular organisms. In particular, gene regulatory mechanisms involving miRNA are critical for both plants and animals. Therefore, understanding why AtXRN4 degrades selected miRNA targets has implications for XRN homologs in many systems. Similarly, PARN and multiple Ccr4p homologs are not present in yeast, in which much of the pioneering work on mRNA degradation has been done, so work on these deadenylases should also be of broad significance for other eukaryotes. Beyond its contributions to basic knowledge, this project will generate data, mutants and other tools that will be made publicly available. Execution of this project will contribute to the education and career development of diverse young scientists including undergraduates and those from a neighboring institution.