FUS Regulates Activity of MicroRNA-Mediated Gene Silencing

The ubiquitin-like protein ubiquilin 2 (UBQLN2) has been genetically and pathologically linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but its normal cellular functions are not well understood. In a search for UBQLN2-interacting proteins, we found an enrichment of stress granule (SG) components, including ALS/FTD-linked heterogeneous ribonucleoprotein fused in sarcoma (FUS). Through the use of an optimized SG detection method, we observed UBQLN2 and its interactors at SGs. A low complexity, Sti1-like repeat region in UBQLN2 was sufficient for its localization to SGs. Functionally, UBQLN2 negatively regulated SG formation. UBQLN2 increased the dynamics of FUS–RNA interaction and promoted the fluidity of FUS–RNA complexes at a single-molecule level. This solubilizing effect corresponded to a dispersal of FUS liquid droplets in vitro and a suppression of FUS SG formation in cells. ALS-linked mutations in UBQLN2 reduced its association with FUS and impaired its function in regulating FUS–RNA complex dynamics and SG formation. These results reveal a previously unrecognized role for UBQLN2 in regulating the early stages of liquid–liquid phase separation by directly modulating the fluidity of protein–RNA complexes and the dynamics of SG formation.

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“…smFRET via TIRF Microscopy. Single-molecule imaging was performed as previously described by Zhang et al (83).…”

Section: Methodsmentioning

confidence: 99%

Ubiquilin 2 modulates ALS/FTD-linked FUS–RNA complex dynamics and stress granule formation

Alexander

Niaki

Zhang

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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102

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“…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). In 2009, mutations in FUS were shown to be causative for Amyotrophic Lateral Sclerosis (ALS) (Kwiatkowski et al, 2009;Vance et al, 2009).…”

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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“…Moreover, recent studies highlighting the interactions of miRISC components with proteins involved in neurodegenerative disorders, such as HTT, FUS, and Ataxin points toward an important role of miRISC composition in the pathology of these disease which should be investigated in detail (Savas et al, 2008;McCann et al, 2011;Zhang T. et al, 2018). Further, the above-mentioned studies showed that mutant versions of HTT and FUS impair miRNA mediated silencing, providing further evidence for the role of miRISC composition in driving neurodegenerative disorders (Savas et al, 2008;Zhang T. et al, 2018). All these studies indicate that it is not miRNA alone, but the composition of miRISC has a great significance in the pathophysiology of disorders of the nervous system.…”

Section: Discussionmentioning

confidence: 99%

“…These RBPs either associate directly with miRISC to influence its function, or they modulate miRISC activity by binding close to the miRNA binding site in the target mRNAs. Proteins like Fragile X Mental Retardation Protein (FMRP), MOV10 Moloney leukemia virus 10 (MOV10), Ataxin 2, and Fused in Sarcoma (FUS) directly associate with core miRISC to influence its downstream effector functions (Banerjee et al, 2009;McCann et al, 2011;Muddashetty et al, 2011;Sudhakaran et al, 2014;Zhang T. et al, 2018) (Figures 1D,E). The association of FMRP with miRISC was earlier contested as one of the report has shown that FMRP does not associate with miRISC and is an important component of stress granules (Didiot et al, 2008).…”

Section: Diverse Mirisc Composition and Its Regulation Brings About Tmentioning

confidence: 99%

The Role of Dynamic miRISC During Neuronal Development

Nawalpuri

Ravindran

Muddashetty

2020

Front. Mol. Biosci.

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Activity-dependent protein synthesis plays an important role during neuronal development by fine-tuning the formation and function of neuronal circuits. Recent studies have shown that miRNAs are integral to this regulation because of their ability to control protein synthesis in a rapid, specific and potentially reversible manner. miRNA mediated regulation is a multistep process that involves inhibition of translation before degradation of targeted mRNA, which provides the possibility to store and reverse the inhibition at multiple stages. This flexibility is primarily thought to be derived from the composition of miRNA induced silencing complex (miRISC). AGO2 is likely the only obligatory component of miRISC, while multiple RBPs are shown to be associated with this core miRISC to form diverse miRISC complexes. The formation of these heterogeneous miRISC complexes is intricately regulated by various extracellular signals and cell-specific contexts. In this review, we discuss the composition of miRISC and its functions during neuronal development. Neurodevelopment is guided by both internal programs and external cues. Neuronal activity and external signals play an important role in the formation and refining of the neuronal network. miRISC composition and diversity have a critical role at distinct stages of neurodevelopment. Even though there is a good amount of literature available on the role of miRNAs mediated regulation of neuronal development, surprisingly the role of miRISC composition and its functional dynamics in neuronal development is not much discussed. In this article, we review the available literature on the heterogeneity of the neuronal miRISC composition and how this may influence translation regulation in the context of neuronal development.

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FUS Regulates Activity of MicroRNA-Mediated Gene Silencing

Cited by 77 publications

References 59 publications

Ubiquilin 2 modulates ALS/FTD-linked FUS–RNA complex dynamics and stress granule formation

Ubiquilin 2 modulates ALS/FTD-linked FUS–RNA complex dynamics and stress granule formation

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

The Role of Dynamic miRISC During Neuronal Development

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