Eukaryotic 3'->5' exonucleolytic activities are essential for ribosomal RNA and snoRNA maturation, mRNA turnover, and other RNA processing events. The multiple 3'->5' exonucleases in eukaryotes exist as a large complex, termed the exosome. Enzymatic activities responsible for various RNA processing events in plant cells remain virtually unknown. This proposal seeks to provide insights into properties and substrates of the exosome in Arabidopsis thaliana. The investigator has cloned and characterized AtRRP41, an Arabidopsis thaliana homologue of an essential subunit of the yeast exosome. Purified recombinant AtRRP41 protein exhibits a highly processive 3'->5' exonucleolytic activity in vitro towards an oligo(A) substrate or polyadenylated mRNA. Exonucleolytic activity of the AtRRP41 protein is strongly inhibited by binding of the poly(A) binding protein to the poly(A) tail of the mRNA substrate. AtRRP41 complements the yeast rrp41/ski6 null mutation. Intriguingly, we find that the AtRRP41 is a hydrolytic exonuclease, whereas its yeast homologue is a phosphorolytic enzyme. These results suggest that it is the role of AtRRP41 in exosome assembly, rather than its enzymatic properties as an uncomplexed protein, that has been conserved over more than a billion years of evolutionary time and is responsible for rescuing the lethality of the yeast rrp41/ski6 null mutation. Consistent with these findings is the fact that AtRRP41 is predominantly found in the cell in a large complex. The long term goal of this project is to elucidate the composition, protein-protein interactions, substrates, regulation and ultimately all cellular functions of the exosome in plants. Specific aims of the project are: 1) to identify the components of the Arabidopsis exosome; 2) to begin dissecting protein-protein interactions of the Arabidopsis exosome subunits; and 3) to identify some of the RNA substrates processed by the Arabidopsis exosome.
This study of the Arabidopsis (thale cress) exosome complex of ribonucleases (enzymes that degrade RNA) is aimed towards a better understanding of plant gene expression at the RNA level, and as such will have broad relevance, ultimately adding to the knowledge base required to optimize the performance of crop plants.
