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

Stefan Reber,Eva Bentmann,Helen Lindsay,Dorothee Dormann,Silvia M L Barabino,Oliver Mühlemann,Brunno Rocha Levone,Michal Domanski,Jonas Mechtersheimer,Marc-David Ruepp,Anny Devoy,Daniel Jutzi

doi:10.1093/nar/gkab582

Abstract

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. The protein Fused in Sarcoma (FUS) undergoes LLPS and mutations in FUS have been causally linked to the motor neuron disease Amyotrophic Lateral Sclerosis (ALS-FUS). LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. However, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. Hence, we developed a method allowing for the purification of LLPS FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome, depending on its biophysical state. While non-LLPS FUS interacts mainly with factors involved in pre-mRNA processing, LLPS FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, also mitochondrial factors are strongly enriched with LLPS FUS, providing a potential explanation for the observed changes in mitochondrial gene expression in mouse models of ALS-FUS. In summary, we present a methodology to investigate the interactomes of phase separating proteins and provide evidence that LLPS shapes the FUS interactome with implications for function and disease.

Highlights

The biophysical process of liquid–liquid phase separation (LLPS) has drawn considerable attention over the last couple of years
Aiming to better understand the functional consequences of Fused in Sarcoma (FUS) phase separation and to identify FUS interactors under LLPS conditions, we developed a method that allows for the purification of phase separated FUS together with its associated proteins and RNAs, and compared these interactors to FUS interactors that were purified under nonLLPS conditions
EGFP-FUS and enhanced green fluorescent protein (eGFP)-FUS P525L, are capable of undergoing LLPS and forming liquid-like compartments in cells, we performed fluorescence recovery after photobleaching (FRAP) on HeLa FUS-KO cells transiently transfected with either FUS WT-GFP or FUS P525L-GFP and photobleached eGFP-FUS granules, and a nearby area to assess fluorescence recovery

Summary

Introduction

The biophysical process of liquid–liquid phase separation (LLPS) has drawn considerable attention over the last couple of years. FUS is a ubiquitously expressed RNA-binding protein that has been implicated in diverse RNA metabolic pathways, such as transcription, pre-mRNA splicing and miRNA processing [11,12,13,14,15,16]. In 2009, mutations in FUS were shown to be causative for Amyotrophic Lateral Sclerosis (ALS) [17,18]. ALS is the most common motor neuron disease in human adults and is characterized by a progressive loss of upper and lower motor neurons, causing paralysis and leading to death [19].

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