Stress granules and neurodegeneration

Wolozin, Benjamin; Ivanov, Pavel

doi:10.1038/s41583-019-0222-5

Cited by 536 publications

(621 citation statements)

References 190 publications

(303 reference statements)

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“…Noteworthy, recruitment of FUS into phase-separated RNP granules, e.g. stress granules , followed by aggregation has been proposed to drive disease (Alberti and Hyman Anthony, 2016;Wolozin and Ivanov, 2019).…”

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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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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“…In addition to mRNAs, SGs are enriched in RNA-binding proteins and translation initiation factors (Nunes et al, 2019). There has been significant effort towards understanding SG composition, material properties, and role in diseases, however, their biological function remains largely unknown (Alberti and Dormann, 2019;Guzikowski et al, 2019;Wolozin and Ivanov, 2019).…”

Section: Introductionmentioning

confidence: 99%

Single-molecule imaging reveals translation of mRNAs localized to stress granules

Matějů

Eichenberger

Eglinger

et al. 2020

Preprint

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Cellular stress leads to reprogramming of mRNA translation and formation of stress granules (SGs), membraneless organelles consisting of mRNA and RNA-binding proteins. Although the function of SGs remains largely unknown, it is widely assumed they contain exclusively nontranslating mRNA. Here we re-examine this hypothesis using single-molecule imaging of mRNA translation in living cells. While our data confirms that non-translating mRNAs are preferentially recruited to SGs, we find unequivocal evidence for translation of mRNA localized to SGs. Our data indicate that SG-associated translation is not rare and that the entire translation cycle (initiation, elongation and termination) can occur for these transcripts. Furthermore, translating mRNAs can be observed transitioning between the cytosol and SGs without changing their translational status. Together, these results argue against a direct role for SGs in inhibition of mRNA translation. observation of biochemical reactions inside and outside of biomolecular condensates in order to understand their biological function. RESULTS Single-molecule imaging reveals SG-associated translationIn order to characterize the relationship between mRNA localization and translation during stress, we combined live-cell imaging of SGs with SunTag-based single-molecule imaging of translation (Pichon et al., 2016;Wang et al., 2016;Wu et al., 2016;Yan et al., 2016). This approach allows visualization of individual reporter mRNAs via MS2 stem-loops and simultaneous visualization of translation via the SunTag, which consists of an array of antibody epitopes that are recognized by genetically-encoded single-chain antibodies (scFv). To accomplish this, we engineered a HeLa cell line stably expressing GFP-tagged scFv and Halo-tagged MS2 coat protein (MCP) . The reporter mRNA encodes the SunTag array in frame with Renilla luciferase, enabling the measurement of protein synthesis by two complementary assays. The reporter mRNA further contains a destabilized FKBP domain to enhance the degradation of the mature protein, thereby facilitating imaging of nascent peptides during translation. To enable visualization of translation during stress, we further placed the 5'UTR of ATF4 in front of the SunTag array ( Figure 1A). This enables the mRNA to be efficiently translated during stress conditions when many other mRNAs are inhibited. The reporter mRNA was integrated into the genome at a site-specific locus and was under the control of a doxycycline-inducible promoter, which enables precise mRNA tracking and minimizes potential artifacts of overexpression. Using luciferase assays, we confirmed that the translation of this ATF4-SunTag reporter is specifically upregulated during arsenite stress and repressed under normal growth conditions ( Figure 1B), as previously reported for endogenous ATF4 (Andreev et al., 2015;Roybal et al., 2005). Importantly, ATF4-SunTag was efficiently translated at an oxidative stress condition (100 µM arsenite) that also triggered robust SG formation in all cells ( Figure 1...

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“…Although the precise functions of SG remain to be determined, they are thought to play a protective role during cellular stress. However, persistent and abnormal SG may nucleate insoluble aggregates that are associated with human neurodegenerative diseases , such as amyotrophic lateral sclerosis (ALS), a neurodegenerative disease of the human motor neuron system (Alberti and Dormann, 2019, Shorter, 2019, Wolozin and Ivanov, 2019.…”

Section: Introductionmentioning

confidence: 99%

“…Intrinsically disordered regions (IDRs) underlie RNA-binding protein capacity to phase separate and condense SG. Accordingly, ALS-causing mutations are prevailing in IDRs and are thought to alter phase separation propensities and SG dynamics (Dormann et al, 2010, Kim et al, 2013, Molliex et al, 2015, Patel et al, 2015, Boeynaems et al, 2017, Hofweber et al, 2018, Elden et al, 2010, Wolozin and Ivanov, 2019. Post-translational modifications (PTMs), are preferentially affecting IDRs (Bah and Forman-Kay, 2016).…”

Section: Introductionmentioning

confidence: 99%

Spatio-temporal Proteomic Analysis of Stress Granule Disassembly Using APEX Reveals Regulation by SUMOylation and Links to ALS Pathogenesis

Siany

Kedersha

Knafo

et al. 2020

Preprint

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Highlights• Multi bait APEX proximity labelling reveals 109 novel SG proteins and two distinct SG substructures.• Proteomic dissection of SG temporal disassembly under basal conditions and with a model of neurodegeneration. SummaryCytoplasmic stress granules (SG) form in response to a variety of cellular stresses by phase-separation of proteins associated with non-translating mRNAs. SG provide insight into the biology of neurodegeneration, including amyotrophic lateral sclerosis (ALS) because they approximate some of the molecular conditions for nucleation of insoluble aggregates in neuropathological inclusions.Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SG undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of SG temporal disassembly sequence, using multi-bait APEX proximity-proteomics. We discover 109 novel SG-proteins and characterize at proteomic resolution two biophysically distinct SG substructures. We further demonstrate that dozens of additional proteins are recruited to SG specifically during disassembly, indicating that it is a highly regulated process. The involved proteins link SG disassembly, to mitochondrial biology and the cytoskeleton. Parallel analysis with C9ORF72-associated dipeptides, which are found in patients with ALS and frontotemporal dementia, demonstrated compositional changes in SG during the course of disassembly and focused our attention on the roles SUMOylation in SG disassembly. We demonstrate that broad SUMOylation of SG-proteins is required for SG disassembly and is impaired by C9ORF72-associated dipeptides, representing an unexplored potential molecular mechanism of neurodegeneration. Altogether, out study fundamentally increases the knowledge about SG composition in human cells by dissecting the SG spatio-temporal proteomic landscape, provides an in-depth resource for future work on SG function and reveals basic and disease-relevant mechanisms of SG disassembly.

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Stress granules and neurodegeneration

Cited by 536 publications

References 190 publications

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

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

Single-molecule imaging reveals translation of mRNAs localized to stress granules

Spatio-temporal Proteomic Analysis of Stress Granule Disassembly Using APEX Reveals Regulation by SUMOylation and Links to ALS Pathogenesis

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