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

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, 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.

ALS-associated FUS mutation reshapes the RNA and protein composition of Stress Granules

“…However, the similar protein population detected does not exclude that FUS P525L could affect SG proteome arrangement. In fact, using published datasets of known FUS protein interactors (42,(44)(45)(46)(47), we found a consistent overlap with already identified SG components. Noteworthy, we observed that some of these FUS protein interactors are more engaged into SG, and their RNA binding ability could contribute to the described transcriptome alterations.…”

“…Noteworthy, we observed that some of these FUS protein interactors are more engaged into SG, and their RNA binding ability could contribute to the described transcriptome alterations. Furthermore, we also observed that the FUS-interacting protein components of SG exhibit a significantly higher potential to form protein-protein physical interactions; this characteristic has been previously documented for the aggregated state of FUS R522G interactors (42). Moreover, the physical interactions considered for this analysis include those involved in the formation of protein complexes (STRING database) and extend beyond contacts simply involving the low-complexity domains (LCDs) of proteins.…”

“…Moreover, we compared the SG proteome in FUS WT condition with other published SG proteomes datasets from HEK293T cells (42) and U2OS cells (17), observing that a consistent portion of the SG localized proteins (77 common core proteins) are shared by all the three systems (Supplementary Fig. 4C).…”

“…However, despite the qualitative similarity in SG protein contents, it is not excluded that the presence of the mutant FUS itself could have an impact on SG proteome organization. Thus, in order to better understand the relationship between FUS protein interactors and the SG proteome, we took advantage of 5 published datasets of FUS protein interactors related to its soluble (44)(45)(46), condensed (47) or aggregated form (42). The latter dataset refers to a mislocalized and aggregated form of FUS carrying the R522G mutation, and displayed the highest amount of FUS protein interactors (488 proteins).…”