Defining the Role of DDX6 in Regulating FUS Condensates

项目总结 融合在肉瘤(FUS)是一种核RNA结合蛋白，经历液-液相分离(LLP)。 当FUS定位错误和/或调节失调时，异常的相分离会导致致病性 与包括肌萎缩侧索硬化症在内的神经退行性疾病有关的固体样聚集体 (ALS)和额颞部痴呆(FTD)。我们最近发现，核糖核酸DDX6的还原 已知的与FUS相互作用的解旋酶在应激过程中显著减少细胞质FUS颗粒的形成 神经母细胞瘤细胞(SH-SY5Y)。我们的初步结果表明：(I)DDX6调节FUS凝析油 数量和大小在体外以浓度依赖的方式，即DDX6促进FUS凝析油的形成 在低浓度下，但在有限水平上限制生长，以及(Ii)DDX6在FUS周围形成一个不连续的环 凝析油。在这些令人兴奋的结果的基础上，我的目标是测试DDX6调节FU的假设 颗粒通过颗粒成核剂和酸洗剂的双重作用。刺激剂是一种颗粒，含有 独特的性质，可以吸附在凝析油表面，提高其流动性，并保持较小的流动性 凝析油大小。根据我们的结果，我们认为DDX6在促进FUS颗粒成核的同时，它的作用 作为Pickering试剂，可保持小液滴尺寸和流动性，从而防止病原性聚集。 此外，我们预测ATP结合和RNA结构将影响FUS颗粒上DDX6的活性。我们 将在三个目标上检验这些预测。AIM 1将利用体外缩合试验来评估相互作用 在DDX6、FUS、RNA和ATP之间进行比较，并确定DDX6是否作为Pickering试剂。AIM 2将利用 生物化学和单分子分析表征分子水平上相互作用的动力学 FUS、DDX6和RNA及其对ATP的依赖。最后，Aim 3将使用基于细胞的方法来研究 调节细胞内DDX6浓度和破坏ATP结合对FUS颗粒形成的影响 野生型和肌萎缩侧索硬化症相关突变。总而言之，这项工作将导致对rna-蛋白质的更深层次的理解。 颗粒调节，对治疗ALS、FTD等神经退行性疾病至关重要。

PROJECT SUMMARY Fused in sarcoma (FUS) is a nuclear RNA binding protein that undergoes liquid-liquid phase separation (LLPS). When mislocalized and/or dysregulated, aberrant phase separation of FUS leads to the formation of pathogenic solid-like aggregates that are implicated in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We have recently discovered that reduction of DDX6, an RNA helicase known to interact with FUS, significantly diminishes cytoplasmic FUS granule formation during stress in neuroblastoma cells (SH-SY5Y). Our preliminary results show that (i) DDX6 modulates FUS condensate number and size in a concentration dependent manner in vitro i.e., DDX6 promotes FUS condensate formation at low concentrations but limits growth above a finite level and (ii) DDX6 forms a discontinuous ring around FUS condensates. Building on these exciting results, I aim to test the hypothesis that DDX6 regulates FUS granules through dual roles as a granule nucleator and Pickering agent. Pickering agents are particles with distinct properties which adsorb to the surface of condensates, promoting their liquidity and maintaining small condensate size. Based on our results, we propose that DDX6 promotes FUS granule nucleation while its role as a Pickering agent maintains small droplet size and liquidity, thus preventing pathogenic aggregation. Additionally, we predict that ATP binding and RNA structure will affect the activity of DDX6 on FUS granules. We will test these predictions in three aims. Aim 1 will utilize in vitro condensation assays to evaluate the interaction between DDX6, FUS, RNA and ATP and to establish whether DDX6 acts as a Pickering agent. Aim 2 will utilize biochemical and single molecule assays to characterize the molecular-level dynamics of the interaction between FUS, DDX6, and RNA, and its dependence on ATP. Finally, aim 3 will use cell-based methods to investigate how tuning intracellular DDX6 concentrations and disrupting ATP binding affects FUS granule formation in wildtype and ALS-associated mutants. Together, this work will lead to a deeper understanding of RNA-protein granule regulation which is of utmost importance to treating neurodegenerative diseases such as ALS and FTD.