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

July 14, 2021

NOMIS researcher Marc-David Ruepp and colleagues have determined the different protein and RNA interactome of soluble and phase-speparated FUS. Their research was published in Nucleic Acids Research on July 7.

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.

Co-IP and purification of LLPS FUS followed by quantitative mass spectrometry and RNA deep sequencing. (A) Experimental workflow. HEK293T cells expressing wild type or P525L eGFP-FUS fusion protein are lysed and subsequently subjected either to a co-IP experiment using anti-GFP nanobodies coupled to magnetic beads (left path) or to eGFP-FUS droplet purification (right path). Droplets are generated through reducing the volume of the lysate and stabilized using the reversible crosslinker formaldehyde. Thereafter, the droplets are purified by fluorescence-activated particle sorting and additional wash steps. (B) Constructs used for co-IP and droplet purification experiments. eGFP fused to FUS including a GSG15 linker between the two proteins. Wild type eGFP-FUS (top right) localizes mainly to the nucleus whereas ALS mutant P525L eGFP-FUS (bottom right) localizes predominantly to the cytoplasm as shown by fluorescence microscopy of transiently transfected HeLa cells counterstained with DAPI. Scale bar = 30 μm. (C) Summary of quantitative mass spectrometry (top) and RNA deep sequencing (bottom) experiments. Shown are numbers for protein and RNA species, which were significantly enriched in co-immunopurification and droplet purification experiments comprising the respective overlap between the two datasets.

Read the Nucleic Acids Research publication: The phase separation-dependent FUS interactome reveals nuclear and cytoplasmic function of liquid–liquid phase separation