Gonzales Fernando A, Zanchin Nilson I T, Luz Juliana S, Oliveira Carla C
Department of Biochemistry, Chemistry Institute, USP, São Paulo, SP, Brazil.
J Mol Biol. 2005 Feb 18;346(2):437-55. doi: 10.1016/j.jmb.2004.11.071. Epub 2004 Dec 21.
In eukaryotes, pre-rRNA processing depends on cis-acting elements and on a large number of non-ribosomal trans-acting factors, including endonucleases and exonucleases, RNA helicases, rRNA modifying enzymes and components of snoRNPs. The exosome is a conserved eukaryotic protein complex containing multiple 3'-5' exonucleases, which has been implicated in pre-rRNA, snoRNA and snRNA processing, as well as in mRNA degradation. In order to identify new proteins involved in rRNA processing, we have screened a yeast two-hybrid cDNA library, to isolate proteins interacting with the exosome subunit Rrp43p. In this screen, a novel nucleolar protein, Nop17p, was identified which also interacts with the box C/D snoRNP protein Nop58p. The NOP17 gene is not essential for cell viability but its deletion causes a temperature-sensitive phenotype. Pre-rRNA processing analyses revealed that rRNA formation is affected in the Deltanop17 strain subjected to the non-permissive temperature, although it is not blocked completely. In addition, primer extension analyses of RNA isolated from Nop17p-depleted cells subjected to the non-permissive temperature indicates that the pre-rRNA is undergoing different modification or degradation processes in these cells as compared to the parental strain. Nop17p was recently described in the same complex as Nop58p and, interestingly, its depletion leads to mislocalization of Nop1p, Nop56p, Nop58p and Snu13p, which are the core proteins of the box C/D ribonucleoprotein (snoRNP), indicating that Nop17p function is required either for nucleolar retention or for the proper assembly of the box C/D snoRNP.
在真核生物中，前体rRNA加工依赖于顺式作用元件和大量非核糖体反式作用因子，包括核酸内切酶和核酸外切酶、RNA解旋酶、rRNA修饰酶以及小核仁核糖核蛋白颗粒（snoRNP）的组分。外切体是一种保守的真核蛋白质复合物，含有多种3'-5'核酸外切酶，它与前体rRNA、snoRNA和snRNA加工以及mRNA降解有关。为了鉴定参与rRNA加工的新蛋白质，我们筛选了一个酵母双杂交cDNA文库，以分离与外切体亚基Rrp43p相互作用的蛋白质。在这个筛选过程中，鉴定出一种新的核仁蛋白Nop17p，它也与C/D框snoRNP蛋白Nop58p相互作用。NOP17基因对细胞活力不是必需的，但其缺失会导致温度敏感型表型。前体rRNA加工分析表明，在非允许温度下的Δnop17菌株中，rRNA的形成受到影响，尽管没有完全阻断。此外，对在非允许温度下从缺失Nop17p的细胞中分离的RNA进行引物延伸分析表明，与亲本菌株相比，这些细胞中的前体rRNA正在经历不同的修饰或降解过程。Nop17p最近被描述为与Nop58p存在于同一复合物中，有趣的是，它的缺失导致C/D框核糖核蛋白（snoRNP）的核心蛋白Nop1p、Nop56p、Nop58p和Snu13p的定位错误，这表明Nop17p的功能对于核仁保留或C/D框snoRNP的正确组装是必需的。
