ALS-linked cytoplasmic FUS assemblies are compositionally different from physiological stress granules and sequester hnRNPA3, a novel modifier of FUS toxicity

Abstract

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, structural differences between mFAs and physiological SGs remain largely unknown therefore it is unclear whether mFAs can functionally substitute for SGs and how they affect cellular stress responses. Here we used affinity purification to isolate mFAs and physiological SGs and compare their protein composition. We found that proteins within mFAs form significantly more physical interactions than those in SGs however mFAs fail to recruit many factors involved in signal transduction. Furthermore, we found 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 mFA-specific protein, hnRNPA3, confirmed its RNA-dependent interaction with FUS and its sequestration into FUS inclusions in cultured cells and in a FUS transgenic mouse model. Silencing of the Drosophila hnRNPA3 ortholog was deleterious and potentiated human FUS toxicity in the retina of transgenic flies. In conclusion, we show that SG-like structures formed by mutant FUS are structurally distinct from SGs, prone to persistence, likely cannot functionally replace SGs, and affect a spectrum of cellular pathways in stressed cells. Results of our study support a pathogenic role for cytoplasmic FUS assemblies in ALS-FUS.

The Enigma of UV Stress Granules: Research Challenges and New Perspectives

“…Identifying the components of UV SGs is a key step to understanding these unusual SGs. Biochemical purification strategies and proximity labeling studies have enabled us to understand the proteomic and transcriptomic landscapes of canonical SGs (Jain et al, 2016; Khong et al, 2017; Markmiller et al, 2018; Namkoong et al, 2018; Padrón et al, 2019; Youn et al, 2019; Marmor-Kollet et al, 2020; Matheny et al, 2021; Vu et al, 2021; An et al, 2022). However, parallel studies on UV SGs, or any non-canonical subtype, remain to be performed.…”

“…Pathological SGs (pSGs) are chronic SGs that form in diseased cells and are hypothesized to seed the accumulation of irreversible and toxic disease aggregates. pSGs have been most closely associated to date with ALS and Alzheimer’s Disease (Vanderweyde et al, 2012, 2016; Ash et al, 2014; Aulas and vande Velde, 2015; Apicco et al, 2018; Zhang et al, 2019; Marmor-Kollet et al, 2020; An et al, 2022). Several excellent reviews have been written on the subject (Wolozin, 2014; Aulas and vande Velde, 2015; Wolozin and Ivanov, 2019), including one within this Research Topic (Rhine et al, 2022).…”

Modulating biomolecular condensates: a novel approach to drug discovery

“…In a recent report, An et al 111 demonstrated that aberrant, persistent pathological stress granules formed by an ALS-associated FUS mutant exhibit different proteomics compared with normal stress granules. These stress granules are characterized by enriched physical interactions between components, consistent with earlier observations that pathological stress granules are less dynamic.…”

Shiftless, a Critical Piece of the Innate Immune Response to Viral Infection

“…A growing number of studies have demonstrated that IFN signaling proteins often re-localize to SG upon viral infection [89,90]. Furthermore, several anti-viral proteins, some of which also ISG, are known constituents of RNP granules including FUS, MOV10, and more recently TDRD3 [90,91]. These observations all raise critical questions regarding SHFL: Does SHFL restrict the expression of select mRNA by re-localizing them to P-bodies as a part of the IFN response?…”