Phillips Seasson, Butler J Scott
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
RNA. 2003 Sep;9(9):1098-107. doi: 10.1261/rna.5560903.
The 3'-5' riboexonuclease Rrp6p, a nuclear component of the exosome, functions with other exosome components to produce the mature 3' ends of 5.8S rRNA, sno- and snRNAs, and to destroy improperly processed precursor (pre)-rRNAs and pre-mRNAs. Rrp6p is a member of the RNase D family of riboexonucleases and displays a high degree of homology with the active site of the deoxyriboexonuclease domain of Escherichia coli DNA polymerase I, the crystal structure of which indicates a two-metal ion mechanism for phosphodiester bond hydrolysis. Mutation of each of the conserved residues predicted to coordinate metal ions in the active site of Rrp6p abolished activity of the enzyme in vitro and in vivo. Complete loss of Rrp6p activity caused by the Y361F and Y361A mutations supports the critical role proposed for the phenolic hydroxyl of Tyr361 in the reaction mechanism. Rrp6p also contains an helicase RNase D C-terminal (HRDC) domain of unknown function that is similar to domains in the Werner's and Bloom's Syndrome proteins. A point mutation in this domain results in Rrp6p that localizes to the nucleus, but fails to efficiently process the 3' ends of 5.8S pre-rRNA and some pre-snoRNAs. In contrast, this mutant retains the ability to degrade rRNA processing intermediates and 3'-extended, poly(A)+ snoRNAs. These findings indicate the potential for independent control of the processing and degradation functions of Rrp6p.
3'-5'核糖核酸外切酶Rrp6p是外切体的一个核组分，它与其他外切体组分共同作用，产生5.8S rRNA、snoRNA和snRNA的成熟3'末端，并降解加工不当的前体（pre）-rRNA和前体mRNA。Rrp6p是核糖核酸外切酶RNase D家族的成员，与大肠杆菌DNA聚合酶I的脱氧核糖核酸外切酶结构域的活性位点具有高度同源性，其晶体结构表明磷酸二酯键水解存在双金属离子机制。预测Rrp6p活性位点中与金属离子配位的每个保守残基发生突变后，在体外和体内均消除了该酶的活性。Y361F和Y361A突变导致Rrp6p活性完全丧失，这支持了Tyr361的酚羟基在反应机制中所起的关键作用。Rrp6p还包含一个功能未知的解旋酶RNase D C末端（HRDC）结构域，该结构域与沃纳综合征和布卢姆综合征蛋白中的结构域相似。该结构域中的一个点突变导致Rrp6p定位于细胞核，但无法有效加工5.8S前体rRNA和一些前体snoRNA的3'末端。相比之下，该突变体保留了降解rRNA加工中间体和3'-延伸的、聚腺苷酸化的snoRNA的能力。这些发现表明Rrp6p的加工和降解功能可能受到独立调控。
