FUS-dependent liquid-liquid phase separation is an early event in double-strand break repair
Preprint posted on August 24, 2020 https://www.biorxiv.org/content/10.1101/798884v2
Categories: biochemistry, cell biology, molecular biology, neuroscience
Context
Liquid-Liquid phase separation (LLPS) coalesces macromolecules from the local cellular milieu to facilitate many biological processes. LLPS has emerged as an important biophysical mechanism to regulate a myriad of the biological process from organizing non-membranous cellular compartments to macromolecules that facilitate cellular and genome function. Notably, in the nucleus, LLPS promotes the spontaneous organization of proteins and nucleic acids with shared functions to facilitate genomic processes like chromatin organization, transcription, RNA processing, DNA replication, and DNA damage response1,2.
In response to DNA damage, cells often commission the proteins available in the local vicinity of the damage for the repair process. This could be the reason for many RNA processing factors to play a crucial role in DNA damage response pathways. Dysregulation of one or many RNA processing factors is known to cause DNA damage and impede genome integrity3. In light of this evidences, the current work investigated the role of LLPS in mediating a DNA/RNA binding protein Fused in sarcoma (FUS) driven DNA damage response.
Key findings
- The investigators made FUS depleted HeLa and SH-SY5Y cells using CRISPR (and shRNA) based technologies to evaluate the role of FUS in the DNA damage response. They found that both cell lines manifested higher levels of DNA damage, as assayed by measuring immunofluorescence staining of DNA damage marker γH2AX. This damage is rescued by ectopic expression of FUS. They also report perturbed DNA damage signaling (assayed by DNA damage marker proteins like γH2AX, 53BP1, pATM, pATR) and low survival rates in FUS depleted cells pharmacologically treated with DNA damaging agents (etoposide and camptothecin).
(1) & (2) FUS recruitment to DNA damage sites precedes SFPQ and its absence strongly reduces SFPQ accumulation. (3) FUS is required for γH2AX nano-foci clustering. Taken and modified directly from Levone B et. al., 2020 under a CC-BY 4.0 international license. - Quantification of γH2AX foci or intensities of γH2AX foci is often used as a read-out to measure DNA damage in human cells. However, in earlier work4, the team demonstrated that γH2AX nano-foci could reveal the 3D organization of γH2AX marked nucleosomes at the DNA damaged chromatin. Building on this, they now show that FUS depletion impedes γH2AX nano-foci clustering that can be rescued by ectopic expression of FUS. Thus, they suggest that FUS is necessary for efficient nano-clustering of γH2AX (and other DNA repair factors) at the DNA damaged chromatin.
- To understand the role of FUS in the early DNA damage response, they analyzed the recruitment kinetics of DNA repair factors at laser-induced microirradiated DNA damage sites. They found that FUS was recruited to DNA damage sites that are prominently marked by γH2AX. FUS recruitment to sites of DNA damage was swift (~5s after damage induction) and preceded SFPQ (~20S after damage induction), an interacting protein of FUS. Furthermore, FUS depletion compromised SFPQ recruitment at DNA damage sites. FUS depletion also modified the recruitment dynamics of other DNA repair factors (KU80, NBS1, 53BP1, BRCA1). Thus, they suggest that FUS acts at early time points of DNA damage response.
- Based on earlier studies, the authors hypothesized whether the ability of FUS and SFPQ to phase separate is necessary for their recruitment to damaged DNA. The investigators demonstrate that LLPS acts as an important driver for FUS and SFPQ mediated DNA damage response by evaluating the recruitment kinetics of FUS and SFPQ to DNA damage sites in the presence of LLPS inhibiting chemicals (1,6-hexanediol and ammonium acetate). They also found that γH2AX and 53BP1 foci were reduced in the presence of LLPS inhibiting chemicals. Furthermore, LLPS-compromised FUS variants did not localize to sites of DNA damage and also hindered SFPQ and KU80 recruitment to the DNA damage sites. Thus, they report that LLPS is crucial to recruit DNA repair factors, and FUS-mediated DNA damage response depends on its LLPS nature.
Conclusion and perspective
Previous reports demonstrate the role of FUS, a DNA/RNA binding protein in DNA damage response5. The current study reports that LLPS is an important property of FUS to act at the onset of damaged DNA, and this is an early event for the repair of damaged DNA. Toxic loss of function mutations in multiple RNA processing genes including FUS causes familial amyotrophic lateral sclerosis (ALS). Moreover, cytoplasmic inclusions of FUS protein aggregates were observed in patients with sporadic ALS5,6. If and how phase separation of toxic FUS variants explain the etiology of FUS-ALS is yet to be elucidated.
Acknowledgments: Thanks to Silvia Barabino and all the authors of this work for their support.
References
- https://doi.org/10.1242/jcs.235093
- https://doi.org/10.1016/j.tibs.2020.06.007
- https://doi.org/10.1016/j.molcel.2009.06.021
- https://doi.org/10.1038/ncomms15760
- https://doi.org/10.1073/pnas.1611673113
- https://doi.org/10.1016/j.brainres.2014.10.009
Posted on: 5th February 2021
doi: https://doi.org/10.1242/prelights.25121
Read preprintSign up to customise the site to your preferences and to receive alerts
Register hereAlso in the biochemistry category:
Also in the cell biology category:
Suppression of Plasmodium MIF-CD74 Signaling Protects Against Severe Malaria
Selected by | Joao Mello-Vieira |
1
Investigating receptor-mediated antibody transcytosis using Blood-Brain Barrier organoid arrays
Selected by | Kristina Kuhbandner |
Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration
Selected by | Jonny Coates |
1
Also in the molecular biology category:
The chromatin, topological and regulatory properties of pluripotency-associated poised enhancers are conserved in vivo
Selected by | Sergio Menchero |
1
Transgenerational Regulation of Sexual Attractiveness in C. elegans Nematodes
Selected by | Yasmin Lau |
The context-dependent, combinatorial logic of BMP signaling
Selected by | Meng Zhu |
1
Also in the neuroscience category:
InsectBrainDatabase - A unified platform to manage, share, and archive morphological and functional data
Selected by | Almut Kelber |
1
Investigating receptor-mediated antibody transcytosis using Blood-Brain Barrier organoid arrays
Selected by | Kristina Kuhbandner |
FUS-dependent liquid-liquid phase separation is an early event in double-strand break repair
Selected by | Ram, Giuseppina D'Alessandro |
1
preListsbiochemistry category:
in theASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar, Ramona Jühlen, Amanda Haage, Laura McCormick, Maiko Kitaoka |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
List by | Dey Lab |
Cellular metabolism
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
List by | Pablo Ranea Robles |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
List by | Sandra Franco Iborra |
Also in the cell biology category:
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
List by | Ana Dorrego-Rivas |
Planar Cell Polarity – PCP
This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.
List by | Ana Dorrego-Rivas |
BioMalPar XVI: Biology and Pathology of the Malaria Parasite
[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria
List by | Dey Lab, Samantha Seah |
1
Cell Polarity
Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.
List by | Yamini Ravichandran |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
List by | Maiko Kitaoka, Madhuja Samaddar, Miguel V. Almeida, Sejal Davla, Jennifer Ann Black, Dey Lab |
3D Gastruloids
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Preprint missing? Don't hesitate to let us know.
List by | Paul Gerald L. Sanchez and Stefano Vianello |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar, Ramona Jühlen, Amanda Haage, Laura McCormick, Maiko Kitaoka |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
List by | Dey Lab |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
List by | Sandra Malmgren Hill |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
List by | Rob Hynds |
Cellular metabolism
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
List by | Pablo Ranea Robles |
BSCB/BSDB Annual Meeting 2019
Preprints presented at the BSCB/BSDB Annual Meeting 2019
List by | Dey Lab |
Biophysical Society Annual Meeting 2019
Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA
List by | Joseph Jose Thottacherry |
ASCB/EMBO Annual Meeting 2018
This list relates to preprints that were discussed at the recent ASCB conference.
List by | Dey Lab, Amanda Haage |
Also in the molecular biology category:
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
List by | Ana Dorrego-Rivas |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar, Ramona Jühlen, Amanda Haage, Laura McCormick, Maiko Kitaoka |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
List by | Rob Hynds |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
List by | Sandra Franco Iborra |
Also in the neuroscience category:
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
List by | Ana Dorrego-Rivas |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar, Ramona Jühlen, Amanda Haage, Laura McCormick, Maiko Kitaoka |
SDB 78th Annual Meeting 2019
A curation of the preprints presented at the SDB meeting in Boston, July 26-30 2019. The preList will be updated throughout the duration of the meeting.
List by | Alex Eve |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
List by | Sandra Malmgren Hill |
Young Embryologist Network Conference 2019
Preprints presented at the Young Embryologist Network 2019 conference, 13 May, The Francis Crick Institute, London
List by | Alex Eve |