The Plant Exosome

核核细胞的3‘-5’外核溶解活性对于核糖体RNA和snoRNA成熟、mRNA转换和其他RNA加工事件至关重要。真核生物中的多个3′-5′外切酶作为一个大的复合体存在，称为外切体。在植物细胞中负责各种RNA加工事件的酶活性实际上仍然未知。本研究旨在深入了解拟南芥外泌体的性质和底物。研究者克隆并鉴定了AtRRP41，这是酵母外体一个重要亚基的拟南芥同源物。纯化的重组AtRRP41蛋白在体外对寡核苷酸(a)底物或聚腺苷化mRNA具有高度的3‘-5’外切活性。通过poly(A)结合蛋白与mRNA底物的poly(A)尾部结合，AtRRP41蛋白的外切核活性被强烈抑制。AtRRP41是酵母rrp41/ski6零突变的补充。有趣的是，我们发现AtRRP41是一种水解核酸外切酶，而它的酵母同源物是一种磷酸化酶。这些结果表明，正是AtRRP41在外显体组装中的作用，而不是其作为一种非复杂蛋白的酶特性，在超过10亿年的进化时间中被保守下来，并负责挽救酵母rrp41/ski6零突变的致病性。与这些发现一致的事实是，AtRRP41主要存在于细胞中的一个大复合体中。该项目的长期目标是阐明植物外泌体的组成、蛋白质-蛋白质相互作用、底物、调控以及最终的所有细胞功能。该项目的具体目标是：1)鉴定拟南芥外泌体的成分；2)开始剖析拟南芥外泌体亚基的蛋白-蛋白相互作用；3)鉴定拟南芥外泌体加工的一些RNA底物。拟南芥（thale cress）核糖核酸酶（降解RNA的酶）外泌体复合体的研究旨在更好地了解RNA水平上的植物基因表达，因此将具有广泛的相关性，最终增加优化作物作物性能所需的知识库。

BelostotskyEukaryotic 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.