Understanding the crosstalk between spatially separated RNP granules during cellular stress responses
Lead Research Organisation:
University of Sheffield
Department Name: Neurosciences
Abstract
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Technical Summary
Phase separation is emerging as a key regulatory principle in eukaryotes, with the assembly and dissolution of membraneless organelles allowing the switch-like regulation of their RNA and protein components activities. Our overarching aim is to understand how RNP granules shape the cellular response to adverse conditions, uncovering how they specialise during stress to exert a specific response and mechanisms of their crosstalk under conditions of spatial segregation.
Given their role in healthy responses and their deregulation in diseases such as viral infections and neurodegeneration, we will use stress granules and paraspeckles as prototypical RNP granules for cytoplasmic and nuclear membraneless organelles. First, we will determine how different stresses trigger the assembly of heterogeneous stress granules and paraspeckles with distinct functional flavours by establishing their structure, dynamics and composition. We will use high-throughput imaging and biochemical isolation of RNP granules coupled to proteomics to define the stress-specific properties of these RNP granules. Next and building on our recent evidence that stress granules can regulate paraspeckles assembly, we will identify the mechanisms responsible for this crosstalk by characterising the signalling pathways and specific proteins involved using both screening and targeted approaches informed by the proteomics. Finally, we will reveal how the assembly of aberrant stress granules impacts on paraspeckles and cellular functions in neurons using models of neurodegenerative diseases; and how the crosstalk between stress granules and paraspeckles contributes to antiviral signalling in infected cells, using viruses known to induce stress granules.
Our study will establish guiding principles for how membraneless organelles specialise and communicate in healthy and pathological conditions, uncovering novel rules of cellular adaptation during stress.
Given their role in healthy responses and their deregulation in diseases such as viral infections and neurodegeneration, we will use stress granules and paraspeckles as prototypical RNP granules for cytoplasmic and nuclear membraneless organelles. First, we will determine how different stresses trigger the assembly of heterogeneous stress granules and paraspeckles with distinct functional flavours by establishing their structure, dynamics and composition. We will use high-throughput imaging and biochemical isolation of RNP granules coupled to proteomics to define the stress-specific properties of these RNP granules. Next and building on our recent evidence that stress granules can regulate paraspeckles assembly, we will identify the mechanisms responsible for this crosstalk by characterising the signalling pathways and specific proteins involved using both screening and targeted approaches informed by the proteomics. Finally, we will reveal how the assembly of aberrant stress granules impacts on paraspeckles and cellular functions in neurons using models of neurodegenerative diseases; and how the crosstalk between stress granules and paraspeckles contributes to antiviral signalling in infected cells, using viruses known to induce stress granules.
Our study will establish guiding principles for how membraneless organelles specialise and communicate in healthy and pathological conditions, uncovering novel rules of cellular adaptation during stress.
Organisations
- University of Sheffield (Lead Research Organisation)
- Murdoch University (Collaboration)
- Ohio State University (Collaboration)
- University of Sheffield (Collaboration)
- University of Groningen (Collaboration)
- University of Essex (Collaboration)
- Goethe University Frankfurt (Collaboration)
- Project MinE (Collaboration)
- KING'S COLLEGE LONDON (Collaboration)
Publications
An H
(2022)
A toolkit for the identification of NEAT1_2/paraspeckle modulators.
in Nucleic acids research
Hodgson R
(2024)
TDP-43 is a Master Regulator of Paraspeckle Condensation
Hodgson RE
(2024)
C9orf72 poly-PR forms anisotropic condensates causative of nuclear TDP-43 pathology.
in iScience
Huang WP
(2024)
Stress-induced TDP-43 nuclear condensation causes splicing loss of function and STMN2 depletion.
in Cell reports
Lang R
(2024)
TDP-43 in nuclear condensates: where, how, and why.
in Biochemical Society transactions
| Description | Biomolecular condensates are emerging as the critical organisers of cellular metabolism and key drivers in numerous diseases. The condensate concept integrates the molecule-scale and systems-biology dimensions and provides a framework for holistic studies of biological phenomena, having led to a paradigm shift in biology in recent years. This project characterised a prototypical cellular biomolecular condensate called the paraspeckle, and its connection with other condensates in the cell, in healthy and diseased cells. In particular, we found that stress inducible nuclear condensates of a protein called TDP-43 critically regulate paraspeckles. This crosstalk becomes affected in neurodegenerative diseases and potentially, viral infections. These insights became possible due to a novel toolkit we built with support from the BBSRC grant, including proteins that undergo condensation in response to blue light. These tools have been made available to the wider research community, through a non-profit repository. The results have been also presented at several scientific conferences at all levels (local, national and international - e.g. Sheffield nucleic acids institute seminar series, Translation UK conference, ALS/MND international symposium and others) and communicated to patients with neurological diseases at an institute open day, thereby engaging a wide range of audiences. This project has provided a springboard for further condensate studies in the lab, Institute, UK-wide and worldwide - via engaging with collaborators. It has also contributed to the career of a postdoctoral researcher who is currently preparing to apply for research fellowship, as well as multiple undergraduate and postgraduate students she has supervised. |
| Exploitation Route | The PI has become involved in establishing a UK-wide network on biomolecular condensates. The inaugural (in-person, 2-day) meeting for this Network took place in January 2025. The PI has since established a group led by UKRI Future Leader Fellows involved in the condensate-applicable research (FLF-CAR) as a key driving force within this Network. A number of activities are currently planned, including an annual symposium, a PI retreat and an ECR summer school by the UK condensate network, with support from FLF-CAR. This will engage and involve new scientists, including ECRs, into condensate research, promoting the key ideas and findings from this project - condensate crosstalk and roles in disease. |
| Sectors | Education Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
| URL | https://ukcondensates.co.uk/ |
| Description | This project has yielded an impact in the educational sector, via the ability of the project staff to contribute to the training of under- and postgraduate students. Furthermore, the staff was engaged in public dissemination of research findings via the Institute open day. In the long-term the discoveries of this project should contribute to the field of synthetic biology and biotechnology (synthetic condensates as bioreactors) as well as drug discovery/pharmaceuticals (condensates as drug targets). |
| First Year Of Impact | 2024 |
| Sector | Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Societal Policy & public services |
| Description | Optogenetic modelling of C9ORF72 DPR pathology in neurons for FTD/ALS research and drug discovery |
| Amount | £67,000 (GBP) |
| Funding ID | ARUK-PPG2023B-007 |
| Organisation | Alzheimer's Research UK |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 08/2024 |
| End | 03/2026 |
| Description | Unravelling the structure and regulation of prototypical membraneless organelles paraspeckles |
| Amount | £44,094 (GBP) |
| Funding ID | BB/X018393/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 05/2023 |
| End | 06/2025 |
| Title | A suite of tools for the identification of small molecule modulators of NEAT1_2/paraspeckles |
| Description | Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) is a long non-coding RNA (lncRNA) with a plethora of physiological and pathological roles. The longer NEAT1 isoform, NEAT1_2, assembles a ribonucleoprotein granule, the paraspeckle. NEAT1_2/paraspeckle dysregulation has been linked to multiple human diseases making it an attractive target for therapy development. However currently the discovery of chemical modulators of NEAT1_2/paraspeckles is hindered by the lack of appropriate tools. To fill this gap, we developed and validated a toolkit comprised of biochemical and cell-based assays for the identification of small molecule NEAT1_2 binders. The NEAT1_2 triple helix stability element was utilised as the target in the biochemical assays, and the cellular assay ('ParaQuant') was based on high-content imaging of NEAT1_2 in fixed cells. As a proof of principle, these assays were used to screen a 1,200-compound FDA-approved drug library and a 170-compound kinase inhibitor library and to confirm the screening hits. The assays are simple to establish, use only commercially-available reagents and are scalable for higher throughput. In particular, ParaQuant is a cost-efficient assay suitable for any cells growing in adherent culture, including motor neurons, and amenable to multiplexing. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2022 |
| Provided To Others? | Yes |
| Impact | Novel tools to guide the search, validation and optimisation of NEAT1_2/paraspeckle-targeted small molecules were developed - methods paper was published in Nucleic Acids Research journal. |
| URL | https://pubmed.ncbi.nlm.nih.gov/36099417/ |
| Title | ImmuCon: in vitro condensate reconstitution for high-resolution imaging |
| Description | ImmCon is an in vitro assay using purified protein for reconstitution of biomolecular condensates and analysis of various factors that affect them. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2023 |
| Provided To Others? | Yes |
| Impact | ImmuCon has been used across several studies, including those with collaborators, and has provided valuable insights into TDP-43 and FUS regulation. |
| URL | https://papers.ssrn.com/sol3/papers.cfm?abstract_id=4721338 |
| Title | In vitro assay for measuring small molecule binding to RNA using FIDA: Flow Induced Dispersion Analysis |
| Description | Assay was developed for NEAT1 binders validaiton using the recently developed Fida technology. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2024 |
| Provided To Others? | No |
| Impact | This assay is being used for compound validation from the MRC/AZ screen of NEAT1 modulators. It will be used in the new grant application for hit-to-lead compound optimisation activities. |
| Title | Optogenetic tools for researching and manipulation of ALS-linked proteins in living cells |
| Description | A panel of genetic constructs for light-driven phase separation/aggregation of ALS-linked proteins (RNA-binding proteins FUS, NONO, TDP-43; and C9ORF72 dipeptide repeat proteins) has been generated and characterised. These constructs have been made available though a non-profit repository Addgene. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2024 |
| Provided To Others? | Yes |
| Impact | These plasmids will be highly instrumental for experiments with spatial and temporal control of physiological and pathological phase separation and now accessible to any researcher via Addgene. |
| URL | https://www.addgene.org/Tatyana_Shelkovnikova/ |
| Description | Analysis of biomolecular condensate dynamics using single molecule imaging |
| Organisation | University of Sheffield |
| Department | Sheffield Institute for Translational Neuroscience (SITraN) |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | This a new collaboration with Dr Alison Twelvetrees. Using her bespoke TIRF imaging setup and protocols, protein dynamics within condensates will be measured in living cells. |
| Collaborator Contribution | Bespoke imaging setup and relevant protocols for condensate labeling and data analysis. |
| Impact | N/A |
| Start Year | 2023 |
| Description | Characterisation of novel nuclear bodies involved in FUS protein autoregulation |
| Organisation | King's College London |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | I have discovered formation of a novel nuclear body by FUS transcripts and now aim to characterise its composition, function and role in FUS autoregulation (including the impact of ALS mutations). |
| Collaborator Contribution | Prof Eugene Makeyev's team developed a novel method for analysis of RNP granule composition by proximity labeling; this method will be applied to characterise the composition of these nuclear bodies. |
| Impact | N/A |
| Start Year | 2022 |
| Description | RNA tracking in live cells using novel chemical probes |
| Organisation | Ohio State University |
| Country | United States |
| Sector | Academic/University |
| PI Contribution | The collaboration is with Prof Dennis Bong whose team is providing non-commercial fluorescent chemical probes for labeling RNA (NEAT1 and C9ORF72) in living cells. These probes are highly complementary to the proposal aims and will allow efficient characterisation of small molecules from project screens in cellular systems. We are providing molecular cloning (plasmid generation) and cell biology expertise within this project. |
| Collaborator Contribution | The collaboration is with Prof Dennis Bong whose team is providing non-commercial fluorescent chemical probes for labeling RNA (NEAT1 and C9ORF72) in living cells. These probes are highly complementary to the proposal aims and will allow efficient characterisation of small molecules from project screens in cellular systems. We are providing molecular cloning (plasmid generation) and cell biology expertise within this project. |
| Impact | None yet, in progress |
| Start Year | 2022 |
| Description | Regulation of FUS splicing |
| Organisation | Goethe University Frankfurt |
| Country | Germany |
| Sector | Academic/University |
| PI Contribution | This partnership led to a new FUS autoregulation mechanism described. My team has uncovered the ability of FUS intron 7 to drive RNA condensation and its potential to be used as a therapeutic agent in ALS-FUS. |
| Collaborator Contribution | Eugene Makeyev's group provided access to a new technology - HyPro-MS, that allows characterising proteomes/interactomes of nuclear bodies and individual RNA species. Michaela Muller-McNicoll provided guidance with experimental design and interpretation (expert in regulation of RNA via intron retention) |
| Impact | BioRxiv preprint: https://www.biorxiv.org/content/10.1101/2025.02.01.633781v1 |
| Start Year | 2023 |
| Description | Regulation of FUS splicing |
| Organisation | King's College London |
| Department | MRC Centre for Developmental Neurobiology |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | This partnership led to a new FUS autoregulation mechanism described. My team has uncovered the ability of FUS intron 7 to drive RNA condensation and its potential to be used as a therapeutic agent in ALS-FUS. |
| Collaborator Contribution | Eugene Makeyev's group provided access to a new technology - HyPro-MS, that allows characterising proteomes/interactomes of nuclear bodies and individual RNA species. Michaela Muller-McNicoll provided guidance with experimental design and interpretation (expert in regulation of RNA via intron retention) |
| Impact | BioRxiv preprint: https://www.biorxiv.org/content/10.1101/2025.02.01.633781v1 |
| Start Year | 2023 |
| Description | Structural variation in NEAT1 in ALS |
| Organisation | Murdoch University |
| Country | Australia |
| Sector | Academic/University |
| PI Contribution | Structural variation in NEAT1_2 was established as potentially contributing to ALS via modulation of TDP-43 binding under stress, in cellular models. This is a collaboration within Sheffield and with external partners. In Sheffield, it is collaboration with Dr J Cooper-Knock and Dr Jenny Lord, as well as Prof Ivana Barbaric. Other partners included Project MinE (Joke van Vugt) and Murdoch University (Ianthe Pitout and Sue Fletcher). |
| Collaborator Contribution | Project MinE database was used for the analysis of NEAT1_2 UG repeat association with ALS severity (ExpansionHunter), with post-analysis done by Dr Cooper-Knock and validation cohort data provided by collaborators at Murdoch University. Subsequently, collaborations were established with Jenny Lord at SITraN (rare diseases informatics) and Ivana Barbaric (neural differentiation) at TUoS School of Biosciences, to examine possible contribution of the developmental component in ALS through impact on NEAT1 expression. |
| Impact | Preprint is published; manuscript in review |
| Start Year | 2023 |
| Description | Structural variation in NEAT1 in ALS |
| Organisation | Project MinE |
| Sector | Charity/Non Profit |
| PI Contribution | Structural variation in NEAT1_2 was established as potentially contributing to ALS via modulation of TDP-43 binding under stress, in cellular models. This is a collaboration within Sheffield and with external partners. In Sheffield, it is collaboration with Dr J Cooper-Knock and Dr Jenny Lord, as well as Prof Ivana Barbaric. Other partners included Project MinE (Joke van Vugt) and Murdoch University (Ianthe Pitout and Sue Fletcher). |
| Collaborator Contribution | Project MinE database was used for the analysis of NEAT1_2 UG repeat association with ALS severity (ExpansionHunter), with post-analysis done by Dr Cooper-Knock and validation cohort data provided by collaborators at Murdoch University. Subsequently, collaborations were established with Jenny Lord at SITraN (rare diseases informatics) and Ivana Barbaric (neural differentiation) at TUoS School of Biosciences, to examine possible contribution of the developmental component in ALS through impact on NEAT1 expression. |
| Impact | Preprint is published; manuscript in review |
| Start Year | 2023 |
| Description | TDP-43 and FUS condensation in health and disease |
| Organisation | University of Essex |
| Country | United Kingdom |
| PI Contribution | This collaboration allowed elucidating the patterns and mechanisms of TDP-43 (co-)condensation. My team has uncovered the disruptive role for TDP-43 on FUS condensation / phase separation, using paraspeckles as models. |
| Collaborator Contribution | Gareth Wright (Essex) provided purified TDP-43 protein fragments and structural biology expertise (AlphaFold predictions). Mark Driver/Patrick Onck (Groningen) performed coarse-grained simulations of TDP-43 - FUS co-condensation. |
| Impact | Manuscript under review |
| Start Year | 2024 |
| Description | TDP-43 and FUS condensation in health and disease |
| Organisation | University of Groningen |
| Country | Netherlands |
| Sector | Academic/University |
| PI Contribution | This collaboration allowed elucidating the patterns and mechanisms of TDP-43 (co-)condensation. My team has uncovered the disruptive role for TDP-43 on FUS condensation / phase separation, using paraspeckles as models. |
| Collaborator Contribution | Gareth Wright (Essex) provided purified TDP-43 protein fragments and structural biology expertise (AlphaFold predictions). Mark Driver/Patrick Onck (Groningen) performed coarse-grained simulations of TDP-43 - FUS co-condensation. |
| Impact | Manuscript under review |
| Start Year | 2024 |
| Description | SITraN Open Day 2024 |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Patients, carers and/or patient groups |
| Results and Impact | SITraN open day that hosted ~100 patients, carers and charity representatives. My group run two stations with hands-on experience (microscopy and molecular biology) - ~6 groups of 5-6 people took part. This was the first open day since the pandemic and was a great success with significant interest from all targeted groups. |
| Year(s) Of Engagement Activity | 2024 |