The phase separation-dependent FUS interactome reveals nuclear and cytoplasmic function of liquid-liquid phase separation

Liquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. One of the best studied proteins undergoing LLPS is Fused in Sarcoma (FUS), a predominantly nuclear RNA-binding protein. Mutations in FUS have been causally linked to Amyotrophic Lateral Sclerosis (ALS), an adult-onset motor neuron disease, and LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. In spite of this, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. In order to study the consequences of LLPS on FUS and its interaction partners, we developed a method that allows for the purification of phase separated FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome of FUS, depending on its biophysical state. While non-phase separated FUS interacts mainly with its well-known interaction partners involved in pre-mRNA processing, phase-separated FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, factors with function in mitochondria are strongly enriched with phase-separated FUS, providing a potential explanation for early changes in mitochondrial gene expression observed in mouse models of ALS-FUS. In summary, we present a methodology that allows to investigate the interactome of phase-separating proteins and provide evidence that LLPS strongly shapes the FUS interactome with important implications for function and disease.

蛋白质与RNA的液-液相分离（Liquid-liquid phase separation, LLPS）已成为无膜细胞器形成的核心驱动因素。这类生物分子凝聚体具备多种生物学功能，且与疾病发生密切相关。研究最为深入的发生LLPS的蛋白之一是肉瘤融合蛋白（Fused in Sarcoma, FUS），这是一类主要定位于细胞核的RNA结合蛋白。FUS的突变与肌萎缩侧索硬化（Amyotrophic Lateral Sclerosis, ALS）——一种成人起病的运动神经元疾病——存在明确的因果关联，而细胞质FUS发生LLPS后聚集的现象被认为是关键的疾病致病机制。尽管如此，目前学界仍不清楚LLPS如何影响细胞内FUS的行为，例如其相互作用组。为了研究LLPS对FUS及其相互作用伴侣的影响，我们开发了一种可从细胞裂解液中纯化出含有FUS的相分离液滴的方法。研究发现，FUS的相互作用组会根据其生物物理状态发生显著改变：未发生相分离的FUS主要与已知的参与前mRNA加工的互作伴侣结合，而发生相分离的FUS则优先结合参与染色质重塑与DNA损伤修复的蛋白。有趣的是，线粒体功能相关因子在相分离FUS中显著富集，这为ALS-FUS小鼠模型中观察到的线粒体基因表达早期变化提供了潜在解释。综上，我们开发了一种可用于研究相分离蛋白相互作用组的方法，并证实LLPS会显著重塑FUS的相互作用组，这一发现对理解其生物学功能与疾病关联具有重要意义。