Villanyi Zoltan, Ribaud Virginie, Kassem Sari, Panasenko Olesya O, Pahi Zoltan, Gupta Ishaan, Steinmetz Lars, Boros Imre, Collart Martine A
Department of Microbiology and Molecular Medicine, University of Geneva, Faculty of Medicine, Geneva, Switzerland; Institute of Genetics and Genomics of Geneva, Geneva, Switzerland.
Department of Biochemistry and Molecular Biology, University of Szeged, Szeged, Hungary.
PLoS Genet. 2014 Oct 23;10(10):e1004569. doi: 10.1371/journal.pgen.1004569. eCollection 2014 Oct.
Recent studies have suggested that a sub-complex of RNA polymerase II composed of Rpb4 and Rpb7 couples the nuclear and cytoplasmic stages of gene expression by associating with newly made mRNAs in the nucleus, and contributing to their translation and degradation in the cytoplasm. Here we show by yeast two hybrid and co-immunoprecipitation experiments, followed by ribosome fractionation and fluorescent microscopy, that a subunit of the Ccr4-Not complex, Not5, is essential in the nucleus for the cytoplasmic functions of Rpb4. Not5 interacts with Rpb4; it is required for the presence of Rpb4 in polysomes, for interaction of Rpb4 with the translation initiation factor eIF3 and for association of Rpb4 with mRNAs. We find that Rpb7 presence in the cytoplasm and polysomes is much less significant than that of Rpb4, and that it does not depend upon Not5. Hence Not5-dependence unlinks the cytoplasmic functions of Rpb4 and Rpb7. We additionally determine with RNA immunoprecipitation and native gel analysis that Not5 is needed in the cytoplasm for the co-translational assembly of RNA polymerase II. This stems from the importance of Not5 for the association of the R2TP Hsp90 co-chaperone with polysomes translating RPB1 mRNA to protect newly synthesized Rpb1 from aggregation. Hence taken together our results show that Not5 interconnects translation and transcription.
最近的研究表明，由Rpb4和Rpb7组成的RNA聚合酶II亚复合物通过在细胞核中与新合成的mRNA结合，并促进其在细胞质中的翻译和降解，将基因表达的核阶段和细胞质阶段联系起来。在这里，我们通过酵母双杂交和免疫共沉淀实验，随后进行核糖体分级分离和荧光显微镜观察，发现Ccr4-Not复合物的一个亚基Not5在细胞核中对Rpb4的细胞质功能至关重要。Not5与Rpb4相互作用；它是Rpb4存在于多核糖体中、Rpb4与翻译起始因子eIF3相互作用以及Rpb4与mRNA结合所必需的。我们发现Rpb7在细胞质和多核糖体中的存在远不如Rpb4显著，并且它不依赖于Not5。因此，Not5依赖性将Rpb4和Rpb7的细胞质功能分开。我们还通过RNA免疫沉淀和天然凝胶分析确定，Not5在细胞质中是RNA聚合酶II共翻译组装所必需的。这源于Not5对于R2TP Hsp90共伴侣与翻译RPB1 mRNA的多核糖体结合以保护新合成的Rpb1不聚集的重要性。因此，综合我们的结果表明，Not5将翻译和转录联系起来。
