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

Abstract

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. The protein Fused in Sarcoma (FUS) undergoes LLPS and mutations in FUS have been causally linked to the motor neuron disease Amyotrophic Lateral Sclerosis (ALS-FUS). LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. However, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. Hence, we developed a method allowing for the purification of LLPS FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome, depending on its biophysical state. While non-LLPS FUS interacts mainly with factors involved in pre-mRNA processing, LLPS FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, also mitochondrial factors are strongly enriched with LLPS FUS, providing a potential explanation for the observed changes in mitochondrial gene expression in mouse models of ALS-FUS. In summary, we present a methodology to investigate the interactomes of phase separating proteins and provide evidence that LLPS shapes the FUS interactome with implications for function and disease.

m6A Modified Short RNA Fragments Inhibit Cytoplasmic TLS/FUS Aggregation Induced by Hyperosmotic Stress

“…Hoell et al reported that the TLS/FUS R521G mutant had a different RNA binding profile compared to WT in HEK293 cells [38], but they also claim that this was probably be due to mislocalization of the TLS/FUS mutant to the cytoplasm. In addition, a recent study revealed that TLS/FUS changes its interactome depending on its state, diffused or aggregated via LLPS [39]. Even in our in vitro experiment, R495X mutant exhibited the lowest specificity to the RNAs examined in this study, which implies NLS of TLS/FUS determines the specificity of RNA binding.…”

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

“… (E) Overlaps between the HEK293 SG core proteomic dataset from the current study and published SG datasets. (F) Overlaps between the mFA core proteome and published human FUS protein interactomes ( Reber et al, 2021 ; Sun et al, 2015 ) and functional assignment of the proteins common for the three datasets ( n = 31). (G) Overlaps between the mFA core proteome and published interactomes of phase-separated/aggregated FUS species ( Kamelgarn et al, 2018 ; Reber et al, 2021 ) and functional assignment of the mFA proteins appearing in at least one of these two published interactomes ( n = 92).…”

“… (F) Overlaps between the mFA core proteome and published human FUS protein interactomes ( Reber et al, 2021 ; Sun et al, 2015 ) and functional assignment of the proteins common for the three datasets ( n = 31). …”

“… (G) Overlaps between the mFA core proteome and published interactomes of phase-separated/aggregated FUS species ( Kamelgarn et al, 2018 ; Reber et al, 2021 ) and functional assignment of the mFA proteins appearing in at least one of these two published interactomes ( n = 92). …”

Tweaking Progranulin Expression: Therapeutic Avenues and Opportunities

“…In mice, similar effects were observed: GRN overexpression could reduce the accumulation of insoluble TDP-43, slow down the axonal loss and improve survival of mutant TDP-43 mice, but was ineffective in mutant SOD1 mice (Herdewyn et al, 2013;Beel et al, 2018). Motor neuron defects in zebrafish caused by FUS mutants or by loss of SMN1, could be rescued by GRN as well (Chitramuthu et al, 2010(Chitramuthu et al, , 2017.…”