Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dundee DD1 5EH, UK.
Institut de Génétique Moléculaire de Montpellier - UMR 5535 CNRS, Université Montpellier 2, 34293 Montpellier Cedex 5, France.
Mol Cell. 2010 Sep 24;39(6):912-924. doi: 10.1016/j.molcel.2010.08.023.
RNA polymerases are key multisubunit cellular enzymes. Microscopy studies indicated that RNA polymerase I assembles near its promoter. However, the mechanism by which RNA polymerase II is assembled from its 12 subunits remains unclear. We show here that RNA polymerase II subunits Rpb1 and Rpb3 accumulate in the cytoplasm when assembly is prevented and that nuclear import of Rpb1 requires the presence of all subunits. Using MS-based quantitative proteomics, we characterized assembly intermediates. These included a cytoplasmic complex containing subunits Rpb1 and Rpb8 associated with the HSP90 cochaperone hSpagh (RPAP3) and the R2TP/Prefoldin-like complex. Remarkably, HSP90 activity stabilized incompletely assembled Rpb1 in the cytoplasm. Our data indicate that RNA polymerase II is built in the cytoplasm and reveal quality-control mechanisms that link HSP90 to the nuclear import of fully assembled enzymes. hSpagh also bound the free RPA194 subunit of RNA polymerase I, suggesting a general role in assembling RNA polymerases.
RNA 聚合酶是关键的多亚基细胞酶。显微镜研究表明，RNA 聚合酶 I 在其启动子附近组装。然而，RNA 聚合酶 II 如何从其 12 个亚基组装仍然不清楚。我们在这里表明，当组装被阻止时，RNA 聚合酶 II 的亚基 Rpb1 和 Rpb3 在细胞质中积累，并且 Rpb1 的核输入需要所有亚基的存在。使用基于 MS 的定量蛋白质组学，我们对组装中间体进行了表征。这些包括含有亚基 Rpb1 和 Rpb8 的细胞质复合物，与 HSP90 共伴侣 hSpagh（RPAP3）和 R2TP/Prefoldin 样复合物相关。值得注意的是，HSP90 活性稳定了细胞质中不完全组装的 Rpb1。我们的数据表明，RNA 聚合酶 II 在细胞质中构建，并揭示了将 HSP90 与完全组装酶的核输入联系起来的质量控制机制。hSpagh 还结合了 RNA 聚合酶 I 的游离 RPA194 亚基，表明其在组装 RNA 聚合酶方面具有普遍作用。
