Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation

Hofweber, Mario; Hutten, Saskia; Bourgeois, Benjamin; Spreitzer, Emil; Niedner-Boblenz, Annika; Schifferer, Martina; Ruepp, Marc‐David; Simons, Mikael; Niessing, Dierk; Madl, Tobias; Dormann, Dorothee

doi:10.1016/j.cell.2018.03.004

Cited by 556 publications

(607 citation statements)

References 71 publications

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“…Indeed, the capacity of several RNA-binding proteins to undergo phase-transition in vitro is inhibited by arginine methylation, e.g. FUS (Hofweber et al, 2018;Qamar et al, 2018) or serine phosphorylation, e.g. TDP-43 .…”

Section: Discussionmentioning

confidence: 99%

Enzymatic degradation of RNA causes widespread protein aggregation in cell and tissue lysates

Aarum

Cabrera

Jones

et al. 2019

Preprint

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Most proteins in cell and tissue lysates are soluble. Here, we show that many of these proteins, including several that are implicated in neurodegenerative diseases, are maintained in a soluble and functional state by association with endogenous RNA, as degradation of RNA invariably leads to protein aggregation. We identify the importance of nucleic acid structure, with single-stranded pyrimidine-rich bulges or loops surrounded by doublestranded regions being particularly efficient in this role, revealing an apparent one-to-one protein-nucleic acid stoichiometry. The relationship of these findings to pathological protein aggregation is suggested by our discovery that protein aggregates isolated from brain tissue from Amyotrophic Lateral Sclerosis patients can be rendered soluble after refolding by both RNA and synthetic oligonucleotides. Together, these findings open new avenues for understanding the mechanism behind protein aggregation and shed light on how certain proteins remain soluble. KEYWORDSmotor neurone disease / protein precipitation / ribonuclease / neurofilament / phase-transition

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Section: Discussionmentioning

confidence: 99%

Enzymatic degradation of RNA causes widespread protein aggregation in cell and tissue lysates

Aarum

Cabrera

Jones

et al. 2019

Preprint

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“…5e). Notably, even under unstressed conditions, P525L FUS containing stress granules were detectable in some cells, presumably due to stress induced through P525L FUS overexpression or reduced chaperoning of FUS-P525L by TNPO1 (Hofweber et al, 2018) and hence enhanced SG recruitment. Furthermore, some wild type FUS expressing cells showed co-localization of FUS together with stress granule markers under stress, likely due to the very high FUS levels leading to cytoplasmic FUS in these cells.…”

Section: Llps Is Not Required For Fus Toxicity In the Cytoplasmmentioning

confidence: 99%

“…LLPS was not only reported to be important for the formation of various membraneless organelles such as nucleoli, P-bodies and stress granules, but also has been implicated in various diseases (Alberti and Carra, 2018;Alberti and Dormann, 2019;Boeynaems et al, 2018). One of the best studied proteins that undergoes LLPS in vitro and in vivo is Fused in Sarcoma (FUS) (Hofweber et al, 2018;Kang et al, 2019;Kato et al, 2012;Monahan et al, 2017;Murray et al, 2017;Patel et al, 2015;Schwartz et al, 2013). FUS is an ubiquitously expressed RNA-binding protein that has been implicated in diverse RNA metabolic pathways, such as transcription, pre-mRNA splicing and miRNA processing (Meissner et al, 2003;Raczynska et al, 2015;Reber et al, 2016;Schwartz et al, 2012;Yu and Reed, 2015;Zhang et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

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

Reber

Lindsay

Devoy

et al. 2019

Preprint

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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...

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“…The interaction between FUS and its import receptor, karyopherin‐ß2 (Karβ2)/importin‐ß2, significantly diminishes FUS aggregation in vitro . The proline/tyrosine‐nuclear localization signal of FUS is frequently mutated in ALS, precluding the FUS/Karβ2 interaction and accelerating FUS aggregation (Figure C). Inhibition of the molecular chaperone, HSP70, also slows the internal dynamics of cellular SGs .…”

Section: Biomolecular Condensates In Neurodegenerationmentioning

confidence: 99%

Biomolecular condensates in neurodegeneration and cancer

Spannl

Tereshchenko

Mastromarco

et al. 2019

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The intracellular environment is partitioned into functionally distinct compartments containing specific sets of molecules and reactions. Biomolecular condensates, also referred to as membrane‐less organelles, are diverse and abundant cellular compartments that lack membranous enclosures. Molecules assemble into condensates by phase separation; multivalent weak interactions drive molecules to separate from their surroundings and concentrate in discrete locations. Biomolecular condensates exist in all eukaryotes and in some prokaryotes, and participate in various essential house‐keeping, stress‐response and cell type‐specific processes. An increasing number of recent studies link abnormal condensate formation, composition and material properties to a number of disease states. In this review, we discuss current knowledge and models describing the regulation of condensates and how they become dysregulated in neurodegeneration and cancer. Further research on the regulation of biomolecular phase separation will help us to better understand their role in cell physiology and disease.

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Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation

Cited by 556 publications

References 71 publications

Enzymatic degradation of RNA causes widespread protein aggregation in cell and tissue lysates

Enzymatic degradation of RNA causes widespread protein aggregation in cell and tissue lysates

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

Biomolecular condensates in neurodegeneration and cancer

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