N6-Methyladenosine RNA Modifications Influence the Stability of FUS-Dependent Biological Condensate Formation.
- 1Department of Chemistry and Biochemistry, Kent State University, Kent, Ohio 44242, United States.
Abstract
Liquid-liquid phase separation (LLPS) is a process in which proteins and nucleic acids organize into dynamic, membrane-less structures within cells. These structures help regulate cellular processes such as gene expression, molecular storage, and stress responses through weak interactions between disordered protein regions and RNA. Emerging evidence highlights a regulatory role for epitranscriptomic modifications, particularly N6-methyladenosine (m⁶A), though the underlying mechanisms remain unclear. Here, we investigated the relationship between m⁶A RNA nucleotide modification and LLPS by identifying proteins that selectively bind methylated RNA. An RNA pull-down assay enriched 106 proteins, including 53 known RNA-binding proteins and several proteins that support LLPS, including FUS (Fused in sarcoma). FUS, a well-characterized phase-separating RNA-binding protein, showed a 2-fold higher binding affinity for m⁶A-modified hairpin RNA than for its unmodified counterpart. Cloud-point analysis and coherent anti-Stokes Raman scattering (CARS) microscopy revealed enhanced thermodynamic stability at higher m⁶A levels in total RNA extracts. Quantitative CARS imaging revealed that, although the overall RNA-to-protein ratio within condensates remains largely unchanged, larger droplets under high-m⁶A conditions exhibit increased RNA content, suggesting that m⁶A strengthens RNA-protein interactions without significantly altering the stoichiometry. Collectively, these findings indicate that while protein-protein interactions may initiate LLPS, m⁶A-modulated RNA-protein interactions govern condensate growth and stability. Furthermore, these results provide a mechanistic insight into how epitranscriptomic modifications regulate biomolecular condensation and influence cellular organization, gene regulation, and disease processes.
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BibTeX
@article{mudiyanselage2026n6methyladenosine,
title = {{N6-Methyladenosine RNA Modifications Influence the Stability of FUS-Dependent Biological Condensate Formation.}},
author = {Chamali Thalagaha Mudiyanselage and Milan Pandey and Sudeshi M Abedeera and Chathurani Ekanayake and Aftab U Mollah and Nadia Anwaar and Arkaprabha Konar and Sanjaya C Abeysirigunawardena},
journal = {ACS bio \& med chem Au},
year = {2026},
month = sep,
doi = {10.1021/acsbiomedchemau.6c00101},
url = {https://europepmc.org/article/MED/42921301},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - N6-Methyladenosine RNA Modifications Influence the Stability of FUS-Dependent Biological Condensate Formation.
AU - Chamali Thalagaha Mudiyanselage
AU - Milan Pandey
AU - Sudeshi M Abedeera
AU - Chathurani Ekanayake
AU - Aftab U Mollah
AU - Nadia Anwaar
AU - Arkaprabha Konar
AU - Sanjaya C Abeysirigunawardena
PY - 2026
DA - 2026/09/15
JO - ACS bio & med chem Au
DO - 10.1021/acsbiomedchemau.6c00101
UR - https://europepmc.org/article/MED/42921301
AB - Liquid-liquid phase separation (LLPS) is a process in which proteins and nucleic acids organize into dynamic, membrane-less structures within cells. These structures help regulate cellular processes such as gene expression, molecular storage, and stress responses through weak interactions between disordered protein regions and RNA. Emerging evidence highlights a regulatory role for epitranscriptomic modifications, particularly N6-methyladenosine (m⁶A), though the underlying mechanisms remain unclear. Here, we investigated the relationship between m⁶A RNA nucleotide modification and LLPS by identifying proteins that selectively bind methylated RNA. An RNA pull-down assay enriched 106 proteins, including 53 known RNA-binding proteins and several proteins that support LLPS, including FUS (Fused in sarcoma). FUS, a well-characterized phase-separating RNA-binding protein, showed a 2-fold higher binding affinity for m⁶A-modified hairpin RNA than for its unmodified counterpart. Cloud-point analysis and coherent anti-Stokes Raman scattering (CARS) microscopy revealed enhanced thermodynamic stability at higher m⁶A levels in total RNA extracts. Quantitative CARS imaging revealed that, although the overall RNA-to-protein ratio within condensates remains largely unchanged, larger droplets under high-m⁶A conditions exhibit increased RNA content, suggesting that m⁶A strengthens RNA-protein interactions without significantly altering the stoichiometry. Collectively, these findings indicate that while protein-protein interactions may initiate LLPS, m⁶A-modulated RNA-protein interactions govern condensate growth and stability. Furthermore, these results provide a mechanistic insight into how epitranscriptomic modifications regulate biomolecular condensation and influence cellular organization, gene regulation, and disease processes.
SN - 2694-2437
ER -