Ahmed Nasiha S, Harrell Lucas M, Wieland Daniel R, Lay Michelle A, Thompson Valery F, Schwartz Jacob C
Department of Molecular and Cellular Biology, The University of Arizona, Tucson, AZ 85719.
Department of Chemistry and Biochemistry, The University of Arizona, Tucson, AZ 85719.
RNA. 2021 May 25;27(8):920-32. doi: 10.1261/rna.078827.121.
Ewing sarcoma is driven by fusion proteins containing a low complexity (LC) domain that is intrinsically disordered and a powerful transcriptional regulator. The most common fusion protein found in Ewing sarcoma, EWS-FLI1, takes its LC domain from the RNA-binding protein EWSR1 (Ewing Sarcoma RNA-binding protein 1) and a DNA-binding domain from the transcription factor FLI1 (Friend Leukemia Virus Integration 1). EWS-FLI1 can bind RNA polymerase II (RNA Pol II) and self-assemble through its low-complexity (LC) domain. The ability of RNA-binding proteins like EWSR1 to self-assemble or phase separate in cells has raised questions about the contribution of this process to EWS-FLI1 activity. We examined EWSR1 and EWS-FLI1 activity in Ewing sarcoma cells by siRNA-mediated knockdown and RNA-seq analysis. More transcripts were affected by the EWSR1 knockdown than expected and these included many EWS-FLI1 regulated genes. We reevaluated physical interactions between EWS-FLI1, EWSR1, and RNA Pol II, and employed a cross-linking based strategy to investigate protein assemblies associated with the proteins. The LC domain of EWS-FLI1 was required for the assemblies observed to form in cells. These results offer new insights into a protein assembly that may enable EWS-FLI1 to bind its wide network of protein partners and contribute to regulation of gene expression in Ewing sarcoma.
尤因肉瘤由含有低复杂性（LC）结构域的融合蛋白驱动，该结构域本质上是无序的，且是一种强大的转录调节因子。在尤因肉瘤中发现的最常见融合蛋白EWS-FLI1，其LC结构域来自RNA结合蛋白EWSR1（尤因肉瘤RNA结合蛋白1），DNA结合结构域来自转录因子FLI1（Friend白血病病毒整合1）。EWS-FLI1可以结合RNA聚合酶II（RNA Pol II）并通过其低复杂性（LC）结构域进行自我组装。像EWSR1这样的RNA结合蛋白在细胞中自我组装或相分离的能力引发了关于这一过程对EWS-FLI1活性贡献的疑问。我们通过小干扰RNA（siRNA）介导的敲低和RNA测序分析，研究了尤因肉瘤细胞中EWSR1和EWS-FLI1的活性。受EWSR1敲低影响的转录本比预期的更多，其中包括许多EWS-FLI1调控的基因。我们重新评估了EWS-FLI1、EWSR1和RNA Pol II之间的物理相互作用，并采用基于交联的策略来研究与这些蛋白质相关的蛋白质组装体。EWS-FLI1的LC结构域是在细胞中观察到形成的组装体所必需的。这些结果为一种蛋白质组装体提供了新的见解，这种组装体可能使EWS-FLI1能够结合其广泛的蛋白质伙伴网络，并有助于调节尤因肉瘤中的基因表达。
