The expression of Rpb10, a small subunit common to RNA polymerases, is modulated by the R3H domain-containing Rbs1 protein and the Upf1 helicase

The biogenesis of eukaryotic RNA polymerases is poorly understood. The present study used a combination of genetic and molecular approaches to explore the assembly of RNA polymerase III (Pol III) in yeast cells. We identified a regulatory link between Rbs1, a Pol III assembly factor, and Rpb10, a small subunit that is common to all three RNA polymerases Overexpression of Rbs1 increased the abundance of both RBP10 mRNA and the Rbp10 protein, which correlated with suppression of Pol III assembly defects. Rbs1 is a poly(A) mRNA-binding protein and mutational analysis identified R3H domain to be required for mRNA interactions and genetic enhancement of Pol III biogenesis. Rbs1 also binds to Upf1 protein, a key component in nonsense-mediated mRNA decay (NMD) and levels of RBP10 mRNA were increased in a upf1∆ strain. Genome-wide RNA binding by Rbs1 was characterized by UV cross-linking based approach (CRAC). We demonstrated that Rbs1 directly binds to the 3’ untranslated regions (3’UTRs) of many mRNAs including transcripts encoding Pol III subunits Rpb10 and Rpc19. We propose that Rbs1 functions by opposing mRNA degradation, at least in part mediated by NMD pathway. Orthologues of Rbs1 protein are present in other eukaryotes, including humans, suggesting that this is a conserved regulatory mechanism.真核RNA聚合酶的生物发生过程目前尚不明晰。本研究结合遗传学与分子生物学手段，探究了酿酒酵母细胞中RNA聚合酶III（Pol III）的组装机制。我们鉴定出Rbs1（一种Pol III组装因子）与Rpb10（三类RNA聚合酶共有的小亚基）之间存在调控关联。Rbs1过表达可同时上调RBP10信使核糖核酸（mRNA）与Rbp10蛋白的表达丰度，该现象与Pol III组装缺陷的抑制效应密切相关。Rbs1是一种结合poly(A)尾mRNA的蛋白，突变分析结果显示，其R3H结构域是介导mRNA结合以及增强Pol III生物发生遗传调控所必需的结构。Rbs1还可结合Upf1蛋白——无义介导的mRNA降解（NMD）通路的关键组分——且在upf1Δ缺失菌株中，RBP10 mRNA的表达水平显著升高。我们采用基于UV交联的技术（CRAC）对Rbs1的全基因组RNA结合谱进行了表征，证实Rbs1可直接结合众多mRNA的3'非翻译区（3’UTRs），其中包括编码Pol III亚基Rpb10与Rpc19的转录本。我们提出，Rbs1通过拮抗mRNA降解发挥功能，该过程至少部分由NMD通路介导。包括人类在内的其他真核生物中均存在Rbs1蛋白的同源物，这表明该调控机制具有进化保守性。