Biochemical and biophysical characterization of the nucleic acid binding properties of the RNA/DNA binding protein EWS.
Biochemical and biophysical characterization of the nucleic acid binding properties of the RNA/DNA binding protein EWS.
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RNA/DNA 结合蛋白 EWS 的核酸结合特性的生化和生物物理表征。
DOI：
10.1002/bip.23536
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发表时间：
2023
期刊：
影响因子：
2.9
通讯作者：
Libich,DavidS
中科院分区：
文献类型：
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作者：
Selig,EmilyE;Bhura,Roohi;White,MatthewR;Akula,Shivani;Hoffman,ReneeD;Tovar,CarmelN;Xu,Xiaoping;Booth,RachellE;Libich,DavidS
EWS 是 RNA/DNA 结合蛋白 FET 家族的成员，可调节核酸代谢的关键阶段。 EWS 包含 N 端低复杂性结构域 (LCD) 和 C 端 RNA 结合结构域 (RBD)。 RBD 进一步分为三个富含 RG 的区域，其侧翼为 RNA 识别基序 (RRM) 和锌指 (ZnF) 结构域。最近，EWS 被证明可以调节尤文肉瘤中的 R 环，尤文肉瘤是一种儿童骨和软组织癌，其中染色体易位将 EWS 的 N 端 LCD 与转录因子 FLI1 的 C 端 DNA 结合域融合。尽管 EWS 被证明可以直接结合 R 环，但结合机制尚未阐明。在当前的研究中，EWS的RBD被分成几个构建体，随后对它们与预期在R环处形成的各种核酸结构的结合进行分析，包括RNA茎环、DNA G四链体和RNA:DNA杂合体。 EWS 以不同的亲和力与所有三种核酸结构相互作用，并且多个结构域有助于结合每种底物。 RRM 和 RG2 区域似乎混杂地结合核酸，而 ZnF 对单链结构表现出更高的选择性。通过这些结果，可以更好地理解 EWS 识别以及 R 环和其他核酸结构结合的结构基础。
EWS is a member of the FET family of RNA/DNA binding proteins that regulate crucial phases of nucleic acid metabolism. EWS comprises an N‐terminal low‐complexity domain (LCD) and a C‐terminal RNA‐binding domain (RBD). The RBD is further divided into three RG‐rich regions, which flank an RNA‐recognition motif (RRM) and a zinc finger (ZnF) domain. Recently, EWS was shown to regulate R‐loops in Ewing sarcoma, a pediatric bone and soft‐tissue cancer in which a chromosomal translocation fuses the N‐terminal LCD of EWS to the C‐terminal DNA binding domain of the transcription factor FLI1. Though EWS was shown to directly bind R‐loops, the binding mechanism was not elucidated. In the current study, the RBD of EWS was divided into several constructs, which were subsequently assayed for binding to various nucleic acid structures expected to form at R‐loops, including RNA stem‐loops, DNA G‐quadruplexes, and RNA:DNA hybrids. EWS interacted with all three nucleic acid structures with varying affinities and multiple domains contributed to binding each substrate. The RRM and RG2 region appear to bind nucleic acids promiscuously while the ZnF displayed more selectivity for single‐stranded structures. With these results, the structural underpinnings of EWS recognition and binding of R‐loops and other nucleic acid structures is better understood.