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RNA-seq to investigate the phase separation-dependent FUS RNA interactome

ABSTRACT: The protein Fused in Sarcoma undergoes liquid-liquid phase separation. To investigate whether this phase transition alters the RNA interactome we purified phase-separated FUS droplets and soluble FUS from HEK 293T cells transfected with GFP-tagged WT FUS or P525L FUS. Phase separated FUS was purified by fluorescence-activated particle sorting (droplets) and soluble FUS by co-Immunoprecipitation (IP) respectively followed by isolation of co-purified RNA. Here we show that phase separation affects the RNA interactome of FUS.

INSTRUMENT(S): Illumina HiSeq 3000

ORGANISM(S): Homo sapiens

SUBMITTER: Stefan Reber

PROVIDER: E-MTAB-8456 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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

Project description: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. One of the best studied proteins undergoing LLPS is Fused in Sarcoma (FUS), a predominantly nuclear RNA-binding protein. Mutations in FUS have been causally linked to Amyotrophic Lateral Sclerosis (ALS), an adult-onset motor neuron disease, and LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. In spite of this, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. In order to study the consequences of LLPS on FUS and its interaction partners, we developed a method that allows for the purification of phase separated FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome of FUS, depending on its biophysical state. While non-phase separated FUS interacts mainly with its well-known interaction partners involved in pre-mRNA processing, phase-separated FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, factors with function in mitochondria are strongly enriched with phase-separated FUS, providing a potential explanation for early changes in mitochondrial gene expression observed in mouse models of ALS-FUS. In summary, we present a methodology that allows to investigate the interactome of phase-separating proteins and provide evidence that LLPS strongly shapes the FUS interactome with important implications for function and disease.

2021-09-08 | PXD015834 | Pride

HspB8 controls molecular aging of FUS condensates by domain-specific protein interactions Part 2

Project description:Biomolecular condensates formed by phase separation offer a confined space where protein-protein interactions (PPIs) facilitate distinct biochemical reactions. As their aberrant behavior is associated with disease, it is crucial to understand the molecular organization of PPIs within condensates. Using quantitative time-resolved crosslinking mass spectrometry (XL-MS) we directly and specifically monitor PPIs and protein dynamics inside condensates formed by the protein fused in sarcoma (FUS) and identify its folded RNA recognition motif (RRM) as a key player of aberrant molecular aging. We find that the chaperone HspB8, but not a disease-associated mutant, prevents FUS aging. In XL-MS and partitioning experiments we find that the disordered region of HspB8 directs its α-crystallin domain (αCD) into FUS droplets where HspB8 prevents aging via condensate-specific αCD–RRM interactions. We propose that chaperones like HspB8 prevent aberrant phase transitions by stabilizing aggregation prone folded domains inside condensates in times of cellular stress.

2021-10-13 | PXD021115 | Pride

Structural insights into protein-RNA interactions within biomolecular condensates using UV cross-linking and mass spectrometry

Project description:UV-light-induced protein-RNA cross-linking followed by MS analysis was used to identify the interaction sites between protein and RNA in different phase-separated and non-phase separated states of complexes involving the following proteins: PTBP1, FUS, and SARS-CoV-2 nucleocapsid protein.

2024-03-06 | PXD046267 | Pride

Identification of FUS RNA targets in HeLa cells

Project description:We identified a landscape of FUS-binding RNA targets in HeLa cells. The majority of the FUS binding sites are in introns of pre-mRNAs and less are in exons and untranslated regions. Significant FUS binding in introns flanking cassette exons, long intron (>100kb) containing transcripts and noncoding RNAs were detected in our study. We specifically determined the function of FUS in regulating the alternative splicing of cassette exons. The top FUS-associated cassette exon is exon 7 of the pre-mRNA of FUS itself. We demonstrated that FUS is a repressor of its own exon 7 splicing. FUS autoregulates its own protein levels by exon 7 alternative splicing and nonsense mediated decay. Moreover, Amyotrophic Lateral Sclerosis (ALS) linked FUS mutants are deficient in FUS autoregulation. CLIP-seq of FUS in HeLa cells

2013-12-16 | E-GEOD-50178 | biostudies-arrayexpress

Mutant FUS triggers age-dependent synaptic impairment in presymptomatic ALS-FUS mice (CLIP-seq)

Project description:FUS is a primarily nuclear RNA-binding protein with important roles in RNA processing and transport. FUS mutations disrupting its nuclear localization characterize a subset of amyotrophic lateral sclerosis (ALS-FUS) patients, through an unidentified pathological mechanism. FUS regulates nuclear RNA, but its role at the synapse is poorly understood. Here, we used super-resolution imaging to determine the physiological localization of extranuclear, neuronal FUS and found it predominantly near the vesicle reserve pool of presynaptic sites. Using CLIP-seq on synaptoneurosome preparations, we identified synaptic RNA targets of FUS that are associated with synapse organization and plasticity. Synaptic FUS was significantly increased in a knock-in mouse model of ALS-FUS, at presymptomatic stages. Despite apparently unaltered synaptic organization, RNA-seq of synaptoneurosomes highlighted age-dependent dysregulation of glutamatergic and GABAergic synapses. Our study indicates that FUS relocalization to the synapse in early stages of ALS-FUS results in synaptic impairment, potentially representing an initial trigger of neurodegeneration.

2021-03-05 | E-MTAB-9211 | biostudies-arrayexpress

FUS KO mRNA sequencing and anti-FUS RNA immunoprecipitation sequencing

Project description:The aim of our study is to identify the role of FUS in shaping the transcriptome. RNA-seq of two FUS KO clones was performed and compared to wt; for each, four replicates were sequenced. RNA molecules associated with the FUS protein were determined by means of a RNA immuno-precipitation, followed by high-throughput sequencing. Total RNA was used as a control. SH-SY5Y cells were used for both experiments. RNA-seq: 4 wt samples, 4 A4 KO samples, 4 A5 KO samples. RIP-seq: 1 input control sample, 3 anti-FUS IP replicates.

2016-06-13 | E-GEOD-71812 | biostudies-arrayexpress

Identification of in vivo, conserved, FUS RNA binding sites and the impact of FUS on the neuronal transcriptome

Project description:FUS, an RNA binding protein was recently implicated in Amyotrophic Lateral Sclerosis (ALS). ALS is a fatal neurodegenerative disease. We report the identification of the conserved neuronal RNA targets of FUS and the assessment of the impact of FUS depletion on the neuronal transcriptome. We identified that FUS regulates splicing of conserved intron containing transcripts. FUS retains or excludes the conserved intron by binding to them. Identification of FUS neuronal targets using normal human brain samples and mouse neurons

2013-01-25 | E-GEOD-43308 | biostudies-arrayexpress

Identification of proteins specifically binding to ac4C RNA probes

Project description:As a newly identified mRNA modification, the regulation of ac4C remains largely unexplored. RNA-binding proteins (RBPs) that specifically binds to ac4C modification and mediate downstream cellular activities (readers) have not been reported yet. We synthesized acetylated and non-acetylated RNA probes by in vitro transcription. The sequences of the probes were segments of FUS and 18s rRNA, which contain ac4C sites as reported. A biotin-RNA pulldown assay and mass spectrometry were performed with HEK 293T cell lysates.

2021-07-20 | PXD027014 | Pride

The oncofusion protein FUS-ERG targets key hematopoietic regulators and modulates the all-trans retinoic acid signaling pathway in t(16;21) acute myeloid leukemia

Project description:Fusion proteins involving the ETS factor ERG have been associated with multiple cancers such as Ewing's sarcoma and prostate cancer. In acute myeloid leukemias harboring t(16;21) another ERG fusion protein is expressed, FUS-ERG. Here, we found that this FUS-ERG oncofusion protein acts in the context of a heptad of proteins (ERG, FLI1, GATA2, LYL1, LNMO2, RUNX1 and TAL1) central to proper expression of genes involved in maintaining a stem cell hematopoietic phenotype. Moreover, in t(16;21) FUS-ERG co-occupies genomic regions bound by the nuclear receptor heterodimer RXR-RARA inhibiting target gene expression and interfering with hematopoietic differentiation. All-Trans Retinoic Acid treatment of t(16;21) cells as well as FUS-ERG knock down alleviate the myeloid differentiation block. Together, the results suggest that FUS-ERG acts as a transcriptional repressor of the retinoic acid signaling pathway. Cell lines were used for RNA extraction for RNA-seq and ChIP experiments for ChIP-seq.

2015-06-23 | E-GEOD-60477 | biostudies-arrayexpress

HspB8 controls hardening and molecular aging of FUS condensates by domain-specific protein interactions Part 1

Project description:Biomolecular condensates formed by phase separation offer a confined space where protein-protein interactions (PPIs) facilitate distinct biochemical reactions. As their aberrant behavior is increasingly associated with disease, it is crucial to understand the molecular organization of PPIs within condensates. Using quantitative and time-resolved crosslinkingmass spectrometry we directly and specifically monitor PPIs and protein dynamics of fused in sarcoma (FUS) condensates and identify its RNA recognition motif (RRM) as a key player of condensate-specific PPIs and aging. We find that the chaperone HspB8, but not a disease-associated mutant, prevents FUS aging. The disordered region of HspB8 directs its α-crystallin domain (αCD) into FUS condensates. Here, RRM unfolding drives aggregation but binding of HspB8 via a droplet-specific αCD – RRM interface significantly delays the onset of aging. We propose that chaperones such as HspB8 prevent aberrant phase transitions by stabilizing aggregation prone RNA binding domains in times of cellular stress.

2021-10-12 | PXD021114 | Pride

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