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

Marmor-Kollet, Hagai; Siany, Aviad; Kedersha, Nancy; Knafo, Naama; Rivkin, Natalia; Danino, Yehuda M.; Moens, Thomas; Olender, Tsviya; Sheban, Daoud; Cohen, Nir; Dadosh, Tali; Addadi, Yoseph; Ravid, Revital; Eitan, Chen; Cohen, Beata Toth; Hofmann, Sarah; Riggs, Claire L.; Advani, Vivek M.; Higginbottom, Adrian; Cooper‐Knock, Johnathan; Hanna, Jacob H.; Merbl, Yifat; Bosch, Ludo Van Den; Anderson, Paul; Ivanov, Pavel; Geiger, Tamar; Hornstein, Eran

doi:10.1016/j.molcel.2020.10.032

Cited by 166 publications

(173 citation statements)

References 91 publications

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“…Thus, insufficient NPC factor recruitment can impair stress signaling pathways, whereas lack of recruitment of the proteasome machinery into mFAs during stress might make them prone to persistence and subsequent conversion into insoluble aggregates. Recently, SGs have been shown to recruit sumoylation factors which facilitate their disassembly, and these factors are depleted from SGs formed in cells accumulating C9ORF72 pathological dipeptides (Marmor-Kollet et al, 2020). Interestingly, the SG but not mFA dataset in our study demonstrated enrichment for sumoylation proteins, which might contribute to impaired disassembly of FUS aggregates.…”

Section: Discussionmentioning

confidence: 99%

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

Litscher

Wei³

et al. 2021

Preprint

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Formation of cytoplasmic RNA-protein structures called stress granules (SGs) is a highly conserved cellular response to stress. Abnormal metabolism of SGs may contribute to the pathogenesis of (neuro)degenerative diseases such as amyotrophic lateral sclerosis (ALS). Many SG proteins are affected by mutations causative of these conditions, including fused in sarcoma (FUS). Mutant FUS variants have high affinity to SGs and also spontaneously form de novo cytoplasmic RNA granules. Mutant FUS-containing assemblies (mFAs), often called 'pathological SGs', are proposed to play a role in ALS-FUS pathogenesis. However, global structural differences between mFAs and physiological SGs remain largely unknown, therefore it is unclear whether and how mFAs may affect cellular stress responses. Here we used affinity purification to characterise the protein and RNA composition of normal SGs and mFAs purified from stressed cells. Comparison of the SG and mFA proteomes revealed that proteasome subunits and certain nucleocytoplasmic transport factors are depleted from mFAs, whereas translation elongation, mRNA surveillance and splicing factors as well as mitochondrial proteins are enriched in mFAs, as compared to SGs. Validation experiments for a hit from our analysis, a splicing factor hnRNPA3, confirmed its RNA-dependent sequestration into mFAs in cells and into pathological FUS inclusions in a FUS transgenic mouse model. Furthermore, silencing of the Drosophila hnRNPA3 ortholog dramatically enhanced FUS toxicity in transgenic flies. Comparative transcriptomic analysis of SGs and mFAs revealed that mFAs recruit a significantly less diverse spectrum of RNAs, including reduced recruitment of transcripts encoding proteins involved in protein translation, DNA damage response, and apoptotic signalling. However mFAs abnormally sequester certain mRNAs encoding proteins involved in stress signalling cascades. Overall, our study establishes molecular differences between physiological SGs and mFAs and identifies the spectrum of proteins, RNAs and respective cellular pathways affected by mFAs in stressed cells. In conclusion, we show that mFAs are compositionally distinct from SGs and that they cannot fully substitute for SG functions while gaining novel, potentially toxic functions in cellular stress response. Results of our study support a pathogenic role for stress-induced cytoplasmic FUS assemblies in ALS-FUS.

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Section: Discussionmentioning

confidence: 99%

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

Litscher

Wei³

et al. 2021

Preprint

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“…In summary, probing the SG interactome at various time points will be required to capture the dynamism of SG-related PPIs. Moreover, the use of multiple bait proteins will be necessary to catalogue the complete SG proteome, as one study has recently reported (20).…”

Section: Discussionmentioning

confidence: 99%

“…There has been considerable effort invested to identify and understand the proteomic (13, 14) and transcriptomic (15, 16) composition of SGs (14, 17–20). Most of these studies have used G3BP1, a core SG protein, to define the SG proteome (19).…”

Section: Introductionmentioning

confidence: 99%

Defining the Caprin-1 interactome in unstressed and stressed conditions

Ghosh

Tran

et al. 2021

Preprint

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Cytoplasmic stress granules (SGs) are dynamic non-membranous foci containing translationally arrested mRNA and RNA binding proteins that form in response to a variety of cellular stressors. SGs may evolve into the cytoplasmic inclusions observed in many neurodegenerative diseases. Recent studies have examined the SG proteome by interrogating the interactome of G3BP1, a core SG protein. To gain further insight into the SG proteome, we employed an immunoprecipitation coupled with mass spectrometry approach of endogenous Caprin-1 in HeLa cells under unstressed or stressed conditions. Overall, we identified ~1,500 proteins that interact with Caprin-1. Interactors under stressed conditions were primarily annotated to the ribosome, spliceosome, and RNA transport pathways. We validated four Caprin-1 interactors that localized to arsenite-induced SGs: ANKHD1, Talin-1, GEMIN5, and SNRNP200. We also validated these stress-induced interactions in SH-SY5Y cells and determined that SNRNP200 also associated with osmotic and thermal induced SGs. Finally, we identified SNRNP200 in cytoplasmic aggregates in ALS spinal cord and motor cortex. Collectively, our findings provide the first description of the Caprin-1 protein interactome, identify novel cytoplasmic SG components, and reveal a SG protein in cytoplasmic aggregates in ALS patients. Proteomic data collected in this study are available via ProteomeXchange with identifier PXD023271.

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“…Very recently, the small ubiquitin-like modifier (SUMO) system and the SUMO-targeted Ub E3 ligase RNF4 were implicated in the process of SG clearance (Keiten-Schmitz et al, 2020;Marmor-Kollet et al, 2020). Therefore, we tested the possibility that the SGassociated Ub conjugates represent, at least in part, mixed SUMO-Ub chains.…”

Section: Sg Association Of Ubiquitin Chains Is Independent Of the Summentioning

confidence: 99%

Comparative profiling of stress granule clearance reveals differential contributions of the ubiquitin system

Tolay

Buchberger

2021

Life Sci. Alliance

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Stress granules (SGs) are cytoplasmic condensates containing untranslated mRNP complexes. They are induced by various proteotoxic conditions such as heat, oxidative, and osmotic stress. SGs are believed to protect mRNPs from degradation and to enable cells to rapidly resume translation when stress conditions subside. SG dynamics are controlled by various posttranslational modifications, but the role of the ubiquitin system has remained controversial. Here, we present a comparative analysis addressing the involvement of the ubiquitin system in SG clearance. Using high-resolution immunofluorescence microscopy, we found that ubiquitin associated to varying extent with SGs induced by heat, arsenite, H2O2, sorbitol, or combined puromycin and Hsp70 inhibitor treatment. SG-associated ubiquitin species included K48- and K63-linked conjugates, whereas free ubiquitin was not significantly enriched. Inhibition of the ubiquitin activating enzyme, deubiquitylating enzymes, the 26S proteasome and p97/VCP impaired the clearance of arsenite- and heat-induced SGs, whereas SGs induced by other stress conditions were little affected. Our data underline the differential involvement of the ubiquitin system in SG clearance, a process important to prevent the formation of disease-linked aberrant SGs.

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

Cited by 166 publications

References 91 publications

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

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

Defining the Caprin-1 interactome in unstressed and stressed conditions

Comparative profiling of stress granule clearance reveals differential contributions of the ubiquitin system

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