Insights into Molecular Diversity within the FET Family: Unraveling Phase Separation of the N-Terminal Low Complexity Domain from RNA-Binding Protein EWS.
Insights into Molecular Diversity within the FET Family: Unraveling Phase Separation of the N-Terminal Low Complexity Domain from RNA-Binding Protein EWS.
复制标题
深入了解 FET 家族内的分子多样性：从 RNA 结合蛋白 EWS 中解开 N 端低复杂性结构域的相分离。
DOI：
10.1101/2023.10.27.564484
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Libich,DavidS
中科院分区：
文献类型：
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作者：
Johnson,CourtneyN;Sojitra,KandarpA;Sohn,ErichJ;Moreno-Romero,AlmaK;Baudin,Antoine;Xu,Xiaoping;Mittal,Jeetain;Libich,DavidS
FET家族蛋白，包括FUS、EWS和TAF 15，是在诸如mRNA成熟、转录调节和DNA损伤反应的过程中起作用的RNA伴侣。这些蛋白具有临床意义：FET蛋白中的染色体重排与尤文家族肿瘤和相关肉瘤有关。此外，FUS和TAF 15的点突变与神经退行性疾病如肌萎缩侧索硬化和额颞叶痴呆有关。融合蛋白EWS：：FLI 1是尤文肉瘤的致病突变，由EWS的低复杂性结构域（LCD）（EWSLCD）与ETS转录因子FLI 1的DNA结合结构域融合的基因组易位引起。这种融合不仅改变了转录程序，而且还阻碍了天然EWS功能，如剪接。然而，本征无序EWSLCD的精确功能仍然是一个积极的研究课题。由于其灵活的性质，EWSLCD可以与自身和其他生物分子形成短暂的相互作用，导致通过相分离形成生物分子缩合物-这一机制被认为是EWS：：FLI 1致癌性的核心。在我们的研究中，我们使用顺磁弛豫增强NMR，分析超离心，光学显微镜，和全原子分子动力学（MD）模拟，以更好地了解EWSLCD的自缔合和相分离的趋势。我们的目的是阐明支持EWSLCD介导的生物分子凝聚的分子事件。我们的NMR数据表明酪氨酸残基主要驱动EWSLCD相分离至关重要的相互作用。此外，酪氨酸残基的较高密度和接近性放大了冷凝物形成的可能性。原子分子动力学模拟和流体动力学实验表明，富含酪氨酸的N和C-末端往往填充紧凑的构象，建立独特的接触网络，这是由一个主要扩展，酪氨酸耗尽，连接区连接。MD模拟提供了关键输入的接触内形成的一个单一的分子（分子内）和内部的凝聚相（分子间），并在蛋白质构象的变化后，冷凝之间的关系。这些结果为FET蛋白的缩合物形成能力提供了更深入的见解，并突出了EWS与其对应物FUS和TAF 15之间独特的结构和功能细微差别。
The FET family proteins, which includes FUS, EWS, and TAF15, are RNA chaperones instrumental in processes such as mRNA maturation, transcriptional regulation, and the DNA damage response. These proteins have clinical significance: chromosomal rearrangements in FET proteins are implicated in Ewing family tumors and related sarcomas. Furthermore, point mutations in FUS and TAF15 are associated with neurodegenerative conditions like amyotrophic lateral sclerosis and frontotemporal lobar dementia. The fusion protein EWS::FLI1, the causative mutation of Ewing sarcoma, arises from a genomic translocation that fuses the low-complexity domain (LCD) of EWS (EWSLCD) with the DNA binding domain of the ETS transcription factor FLI1. This fusion not only alters transcriptional programs but also hinders native EWS functions like splicing. However, the precise function of the intrinsically disordered EWSLCD is still a topic of active investigation. Due to its flexible nature, EWSLCD can form transient interactions with itself and other biomolecules, leading to the formation of biomolecular condensates through phase separation – a mechanism thought to be central to the oncogenicity of EWS::FLI1. In our study, we used paramagnetic relaxation enhancement NMR, analytical ultracentrifugation, light microscopy, and all-atom molecular dynamics (MD) simulations to better understand the self-association and phase separation tendencies of EWSLCD. Our aim was to elucidate the molecular events that underpin EWSLCD-mediated biomolecular condensation. Our NMR data suggest tyrosine residues primarily drive the interactions vital for EWSLCD phase separation. Moreover, a higher density and proximity of tyrosine residues amplify the likelihood of condensate formation. Atomistic MD simulations and hydrodynamic experiments revealed that the tyrosine-rich N and C-termini tend to populate compact conformations, establishing unique contact networks, that are connected by a predominantly extended, tyrosine-depleted, linker region. MD simulations provide critical input on the relationship between contacts formed within a single molecule (intramolecular) and inside the condensed phase (intermolecular), and changes in protein conformations upon condensation. These results offer deeper insights into the condensate-forming abilities of the FET proteins and highlights unique structural and functional nuances between EWS and its counterparts, FUS and TAF15.