Thompson Valery F, Victor Rachel A, Morera Andres A, Moinpour Mahta, Liu Meilani N, Kisiel Conner C, Pickrel Kaitlyn, Springhower Charis E, Schwartz Jacob C
Biochemistry. 2018 Dec 26;57(51):7021-7032. doi: 10.1021/acs.biochem.8b01097. Epub 2018 Dec 11.
Purified recombinant FUsed in Sarcoma (FUS) assembles into an oligomeric state in an RNA-dependent manner to form large condensates. FUS condensates bind and concentrate the C-terminal domain of RNA polymerase II (RNA Pol II). We asked whether a granule in cells contained FUS and RNA Pol II as suggested by the binding of FUS condensates to the polymerase. We developed cross-linking protocols to recover protein particles containing FUS from cells and separated them by size exclusion chromatography. We found a significant fraction of RNA Pol II in large granules containing FUS with diameters of >50 nm or twice that of the RNA Pol II holoenzyme. Inhibition of transcription prevented the polymerase from associating with the granules. Altogether, we found physical evidence of granules containing FUS and RNA Pol II in cells that possess properties comparable to those of in vitro FUS condensates.
纯化的重组肉瘤融合蛋白（FUS）以RNA依赖的方式组装成寡聚状态，形成大的凝聚物。FUS凝聚物结合并浓缩RNA聚合酶II（RNA Pol II）的C末端结构域。我们询问细胞中的颗粒是否如FUS凝聚物与聚合酶的结合所提示的那样含有FUS和RNA Pol II。我们开发了交联方案，从细胞中回收含有FUS的蛋白质颗粒，并通过尺寸排阻色谱法对其进行分离。我们发现，在直径大于50 nm或RNA Pol II全酶直径两倍的含有FUS的大颗粒中，有相当一部分RNA Pol II。转录抑制阻止了聚合酶与颗粒结合。总之，我们在细胞中发现了含有FUS和RNA Pol II的颗粒的物理证据，这些颗粒具有与体外FUS凝聚物相当的特性。
