The Plant Exosome

Belostotsky真核生物的3 '-5'核酸外切活性对于核糖体RNA和snoRNA成熟、mRNA周转和其他RNA加工事件是必不可少的。真核生物中的多个3 '-5'外切核酸酶作为大的复合物存在，称为外泌体。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。本研究旨在为拟南芥外泌体的性质和底物提供新的认识。研究人员已经克隆并表征了AtRRP 41，这是酵母外泌体的一个必需亚基的拟南芥同源物。纯化的重组AtRRP 41蛋白在体外对寡聚（A）底物或聚腺苷酸化的mRNA表现出高度进行性的3 '-5'核酸外切活性。AtRRP 41蛋白的核酸外切活性被poly（A）结合蛋白与mRNA底物的poly（A）尾的结合强烈抑制。AtRRP 41补充酵母rrp 41/ski 6无效突变。有趣的是，我们发现AtRRP 41是一种水解核酸外切酶，而它的酵母同源物是一种磷酸解酶。这些结果表明，AtRRP 41在外泌体组装中的作用，而不是其作为未复合蛋白的酶促特性，在超过10亿年的进化时间中保持不变，并且负责挽救酵母rrp 41/ski 6无效突变的致命性。与这些发现一致的是，AtRRP 41主要存在于细胞中的大复合物中。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、调控以及最终所有的细胞功能。该项目的具体目标是：1）鉴定拟南芥外泌体的组成部分; 2）开始解剖拟南芥外泌体亚基的蛋白质-蛋白质相互作用;以及3）鉴定由拟南芥外泌体加工的一些RNA底物。（塔勒菜）核糖核酸酶外泌体复合物（降解RNA的酶）旨在更好地理解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.