Proteomic analysis of FUS interacting proteins provides insights into FUS function and its role in ALS

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease. Mutations in the Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS) gene cause a subset of familial ALS cases and are also implicated in sporadic ALS. FUS is typically localized to the nucleus. The ALS-related FUS mutations cause cytoplasmic mis-localization and the formation of stress granule-like structures. Abnormal cytoplasmic FUS localization was also found in a subset of frontotemporal dementia (FTLD) cases without FUS mutations. To better understand the function of FUS, we performed wild-type and mutant FUS pull-downs followed by proteomic identification of the interacting proteins. The FUS interacting partners we identified are involved in multiple pathways, including chromosomal organization, transcription, RNA splicing, RNA transport, localized translation, and stress response. FUS interacted with hnRNPA1 and Matrin-3, RNA binding proteins whose mutations were also reported to cause familial ALS, suggesting that hnRNPA1 and Matrin-3 may play common pathogenic roles with FUS. The FUS interactions displayed varied RNA dependence. Numerous FUS interacting partners that we identified are components of exosomes. We found that FUS itself was present in exosomes, suggesting that the secretion of FUS might contribute to the cell-to-cell spreading of FUS pathology. FUS interacting proteins were sequestered into the cytoplasmic mutant FUS inclusions that could lead to their mis-regulation or loss of function, contributing to ALS pathogenesis. Our results provide insights into the physiological functions of FUS as well as important pathways where mutant FUS can interfere with cellular processes and potentially contribute to the pathogenesis of ALS.

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

“…Interestingly, we (this study, see Fig. 2 (PARP1) and Supplementary Table 1) and others (Blokhuis et al, 2016;Bosco et al, 2010;Kamelgarn et al, 2016;Sun et al, 2015) reported interactions between FUS and different stress granule markers. Hence, it is conceivable that LLPS-deficient FUS sequesters factors required for the formation of stress granules and thus prevents the formation of stress granules during stress.…”

“…Nevertheless, many FUS interactors are detected under both conditions but show a clear preference for either dispersed/soluble or phase separated FUS.. Importantly, several proteins enriched with phase separated FUS, such as DDX3X, DHX9, FMR1, TIAR-1 and SMN1 (compare Supplementary Table 1), have already been observed by others to co-localize with FUS into cytoplasmic granules (Blokhuis et al, 2016;Bosco et al, 2010;Groen et al, 2013;Kamelgarn et al, 2016). Moreover, proteins found in nuclear granules, specifically paraspeckles (Shelkovnikova et al, 2014) and transcription-dependent granules containing FUS and RNA Pol II (Thompson et al, 2018) are highly enriched under LLPS conditions compared to non-LLPS conditions (compare data in Supplementary Table 1).…”

“…This indicates different affinities, but not necessarily mutually exclusive binding to FUS, depending on its biophysical state. We further analysed proteins co-purified with FUS under LLPS and non-LLPS conditions, comparing them to previously reported FUS interactors (Blokhuis et al, 2016;Chi et al, 2018;Hein et al, 2015;Kamelgarn et al, 2016;Reber et al, 2016;Sun et al, 2015;Wang et al, 2015). Interestingly, only 23.8 % of the LLPS-dependent FUS interactors have been previously reported, whereas 47.2 % of the co-IPed FUS interactors have already been reported by these previous studies.…”

Matrin3 binds directly to intronic pyrimidine‐rich sequences and controls alternative splicing

“…; Kamelgarn et al . ; Yamaguchi & Takanashi ), future studies should address how Matrin3 and TDP‐43 or FUS coordinately regulate alternative splicing and how disease‐associated point mutations in MATR3 affect binding to TDP‐43 or FUS in terms of the elucidation of disease pathogenesis.…”

“…It remains undetermined whether disease-associated point mutations in MATR3, which are located outside the RRMs, affect the regulation of splicing by Matrin3. Given that dysregulation of splicing is induced by ALS-linked point mutations in TARDBP and FUS (Arnold et al 2013;Reber et al 2016) and that Matrin3 associates with TDP-43 and FUS (Ling et al 2010;Salton et al 2011;Johnson et al 2014;Kamelgarn et al 2016;Yamaguchi & Takanashi 2016), future studies should address how Matrin3 and TDP-43 or FUS coordinately regulate alternative splicing and how diseaseassociated point mutations in MATR3 affect binding to TDP-43 or FUS in terms of the elucidation of disease pathogenesis.…”