Departamento de Biología Experimental, Facultad de Ciencias Experimentales, Universidad de Jaén, Jaén, Spain.
PLoS Genet. 2013;9(2):e1003297. doi: 10.1371/journal.pgen.1003297. Epub 2013 Feb 14.
The unconventional prefoldin URI/RMP, in humans, and its orthologue in yeast, Bud27, have been proposed to participate in the biogenesis of the RNA polymerases. However, this role of Bud27 has not been confirmed and is poorly elucidated. Our data help clarify the mechanisms governing biogenesis of the three eukaryotic RNA pols. We show evidence that Bud27 is the first example of a protein that participates in the biogenesis of the three eukaryotic RNA polymerases and the first example of a protein modulating their assembly instead of their nuclear transport. In addition we demonstrate that the role of Bud27 in RNA pols biogenesis depends on Rpb5. In fact, lack of BUD27 affects growth and leads to a substantial accumulation of the three RNA polymerases in the cytoplasm, defects offset by the overexpression of RPB5. Supporting this, our data demonstrate that the lack of Bud27 affects the correct assembly of Rpb5 and Rpb6 to the three RNA polymerases, suggesting that this process occurs in the cytoplasm and is a required step prior to nuclear import. Also, our data support the view that Rpb5 and Rpb6 assemble somewhat later than the rest of the complexes. Furthermore, Bud27 Rpb5-binding but not PFD-binding domain is necessary for RNA polymerases biogenesis. In agreement, we also demonstrate genetic interactions between BUD27, RPB5, and RPB6. Bud27 shuttles between the nucleus and the cytoplasm in an Xpo1-independent manner, and also independently of microtubule polarization and possibly independently of its association with the RNA pols. Our data also suggest that the role of Bud27 in RNA pols biogenesis is independent of the chaperone prefoldin (PFD) complex and of Iwr1. Finally, the role of URI seems to be conserved in humans, suggesting conserved mechanisms in RNA pols biogenesis.
非传统的前折叠蛋白 URI/RMP（在人类中）及其在酵母中的同源物 Bud27，被提议参与 RNA 聚合酶的生物发生。然而，Bud27 的这种作用尚未得到证实，也未得到充分阐明。我们的数据有助于阐明调控三种真核 RNA 聚合酶生物发生的机制。我们提供的证据表明，Bud27 是第一个参与三种真核 RNA 聚合酶生物发生的蛋白质的例子，也是第一个调节它们的组装而不是它们的核转运的蛋白质的例子。此外，我们证明了 Bud27 在 RNA 聚合酶生物发生中的作用依赖于 Rpb5。事实上，缺乏 BUD27 会影响生长并导致三种 RNA 聚合酶在细胞质中大量积累，这些缺陷可以通过 RPB5 的过表达来弥补。支持这一点，我们的数据表明 Bud27 的缺乏会影响 Rpb5 和 Rpb6 与三种 RNA 聚合酶的正确组装，这表明该过程发生在细胞质中，并且是核输入之前的必需步骤。此外，我们的数据还支持这样一种观点，即 Rpb5 和 Rpb6 的组装比其他复合物稍晚。此外，Bud27 与 Rpb5 结合但不与 PFD 结合的结构域对于 RNA 聚合酶的生物发生是必需的。一致地，我们还证明了 BUD27、RPB5 和 RPB6 之间存在遗传相互作用。Bud27 以依赖于 Xpo1 的方式在核和细胞质之间穿梭，并且还独立于微管极化，可能独立于其与 RNA pol 的结合。我们的数据还表明，Bud27 在 RNA 聚合酶生物发生中的作用独立于伴侣蛋白前折叠蛋白（PFD）复合物和 Iwr1。最后，URI 的作用在人类中似乎是保守的，这表明在 RNA 聚合酶生物发生中存在保守的机制。
