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

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. 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.Wang et al., 2018) and the wash volumes applied during the co-IP exceeded the volume applied to analyse the cell lysates in Supplementary Fig. 1b, LLPS of FUS during co-IP conditions is limited to the minimum or even completely prevented. A pulldown of FLAG-eGFP served as control IP. Successful purification of the bait from droplet purifications and co-IPs were verified by SDS-PAGE followed by silver staining or western blotting, respectively ( Supplementary Fig. 2). Proteins and RNAs purified from co-IP and droplet purification experiments were analysed by means of quantitative mass spectrometry or RNA deep sequencing, respectively. Interestingly, the wild type and P525L FUS protein and RNA interactomes (under the same experimental conditions) were mostly identical ( Supplementary Fig. 3), which is why wild type and P525L interactomes from the same experimental conditions were pooled, in order to identify the most robust FUS interactors under LLPS and non-LLPS conditions. We identified 238 proteins interacting with FUS under LLPS conditions and 360 under non-LLPS conditions. 102 proteins were present in both datasets (independent of either biophysical state), resulting in 136 proteins specific to the LLPS condition. The observation that several proteins and RNAs preferentially interacted with FUS under LLPS conditions ( Fig. 1c and Supplementary Table 1) indicates th...

Compositional analysis of ALS-linked stress granule-like structures reveals factors and cellular pathways dysregulated by mutant FUS under stress

“…We found that several classes of proteins are abnormally enriched in mFAs, as compared to SGs, including mitochondrial proteins, translation and mRNA surveillance pathway factors, and components of the spliceosome, which is in line with reports by others. Enrichment of mFAs in mitochondrial proteins is consistent with a previous finding that FUS interacts with mitochondria (Deng et al, 2015), and a recent report of specific interaction of phase-separated FUS with mitochondrial proteins and entrapment of mitochondrial ribosomal RNAs in these aggregated FUS fractions (Reber et al, 2019). Binding of translation factors by mutant aggregated FUS was also recently demonstrated (Kamelgarn et al, 2018), and the effect of FUS aggregates on translation was highlighted in several studies (Kamelgarn et al, 2018; Lopez-Erauskin et al, 2018; Scekic-Zahirovic et al, 2016; Yasuda et al, 2013).…”

“…Our data suggest that the majority of pathways dysregulated by mutant FUS under basal conditions, e.g. protein translation, RNA quality control, mitochondrial function, splicing, DNA damage repair and chromatin remodeling (Kamelgarn et al, 2018; Reber et al, 2019), are also affected under stress. However, our approach allowed identification of new pathways and factors dysregulated by mutant FUS assemblies at the protein and RNA level specifically under stress, first of all, proteasome clearance of misfolded proteins/aggregates (at the protein level) and cellular stress/apoptotic signaling (at the RNA level).…”

“…In a recent report, FUS aggregates were captured using a membrane filtration protocol with subsequent proteomic analysis (Kamelgarn et al, 2018). In another recent study, condensed FUS droplets from cell lysates were crosslinked, FACS-sorted and immunoprecipitated, with proteomic and transcriptomic analyses carried out on pulled WT/mutant FUS preparations separately for soluble and phase-separated fractions (Reber et al, 2019). These studies identified core cellular pathways regulated by FUS protein and confirmed that some of them, such as translation and NMD, are compromised in mutant FUS expressing cells.…”

FUS-dependent loading of SUV39H1 to OCT4 pseudogene-lncRNA programs a silencing complex with OCT4 promoter specificity

“…In addition, we did not find SUV39H1 peptides in our mass spectrometry data from mOct4P4 -24xMS2 lncRNA pull down experiments (Supplementary Data 1 ). This is in line with a lack of SUV39H1 in published data on the FUS interacting proteome 34 – 38 . We conclude that direct SUV39H1–FUS interaction is not a pre-requisite for silencing complex formation.…”

Phase Separation and Neurodegenerative Diseases: A Disturbance in the Force

“…Examples of this case may be TDP-43 and FUS, shell components of paraspeckles, to which they are recruited by direct binding to NEAT1 (Lagier-Tourenne et al, 2012;Modic et al, 2019;West et al, 2016). Moreover, both TDP-43 and FUS mostly undergo physiological LLPS in the nucleus (Gasset-Rosa et al, 2019;Maharana et al, 2018;Reber et al, 2019), where they bind their specific pre-mRNA targets on primarily intronic sites (Lagier-Tourenne et al, 2012;.…”