Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York, United States of America.
PLoS One. 2013 May 8;8(5):e63879. doi: 10.1371/journal.pone.0063879. Print 2013.
Unconventional prefoldin RPB5 Interactor (URI) was identified as a transcriptional repressor that binds RNA polymerase II (pol II) through interaction with the RPB5/POLR2E subunit. Despite the fact that many other proteins involved in transcription regulation have been shown to interact with URI, its nuclear function still remains elusive. Previous mass spectrometry analyses reported that URI is part of a novel protein complex called R2TP/prefoldin-like complex responsible for the cytoplasmic assembly of RNA polymerase II. We performed a mass spectrometry (MS)-based proteomic analysis to identify nuclear proteins interacting with URI in prostate cells. We identified all the components of the R2TP/prefoldin-like complex as nuclear URI interactors and we showed that URI binds and regulates RPB5 protein stability and transcription. Moreover, we validated the interaction of URI to the P53 and DNA damage-Regulated Gene 1 (PDRG1) and show that PDRG1 protein is also stabilized by URI binding. We present data demonstrating that URI nuclear/cytoplasmic shuttling is affected by compounds that stall pol II on the DNA (α-amanitin and actinomycin-D) and by leptomycin B, an inhibitor of the CRM1 exportin that mediates the nuclear export of pol II subunits. These data suggest that URI, and probably the entire R2TP/prefoldin-like complex is exported from the nucleus through CRM1. Finally we identified putative URI sites of phosphorylation and acetylation and confirmed URI sites of post-transcriptional modification identified in previous large-scale analyses the importance of which is largely unknown. However URI post-transcriptional modification was shown to be essential for URI function and therefore characterization of novel sites of URI modification will be important to the understanding of URI function.
非常规前体蛋白 RPB5 相互作用因子（URI）被鉴定为一种转录抑制剂，它通过与 RPB5/POLR2E 亚基相互作用来结合 RNA 聚合酶 II（pol II）。尽管已经证明许多其他参与转录调控的蛋白质与 URI 相互作用，但它的核功能仍然难以捉摸。之前的质谱分析报告称，URI 是一种称为 R2TP/前体蛋白样复合物的新型蛋白质复合物的一部分，该复合物负责 RNA 聚合酶 II 的细胞质组装。我们进行了基于质谱（MS）的蛋白质组学分析，以鉴定与前列腺细胞中的 URI 相互作用的核蛋白。我们鉴定了 R2TP/前体蛋白样复合物的所有成分都是核 URI 相互作用因子，并且我们表明 URI 结合并调节 RPB5 蛋白稳定性和转录。此外，我们验证了 URI 与 P53 和 DNA 损伤调节基因 1（PDRG1）的相互作用，并表明 PDRG1 蛋白也被 URI 结合稳定。我们提供的数据表明，URI 的核/细胞质穿梭受到阻止 pol II 在 DNA 上停滞的化合物（α-鹅膏蕈碱和放线菌素-D）以及莱普霉素 B 的影响，莱普霉素 B 是一种 CRM1 输出蛋白抑制剂，介导 pol II 亚基的核输出。这些数据表明，URI，可能还有整个 R2TP/前体蛋白样复合物，通过 CRM1 从细胞核输出。最后，我们确定了假定的 URI 磷酸化和乙酰化位点，并确认了先前大规模分析中鉴定的 URI 转录后修饰位点的重要性，而这些修饰位点的重要性在很大程度上是未知的。然而，URI 的转录后修饰对于 URI 的功能至关重要，因此，对 URI 修饰的新型位点的特征描述对于理解 URI 的功能将是重要的。
