Nicolas Lux Fawzi

Fused in sarcoma (FUS) is an RNA‐binding protein that undergoes phase separation with RNA and other cellular components, forming ribonucleoprotein (RNP) granules. While recent advances in the study of biomolecular phase separation have focused on protein–protein interactions, information on the molecular details of protein–RNA interactions within condensates remains limited. Here, we demonstrate how RNA modulates the phase separation of the low‐complexity (LC) and arginine–glycine–glycine motif (RGG1) domains of FUS—low RNA concentrations enhance protein phase separation and excess RNA disrupts it. Integrating biochemical assays, NMR spectroscopy, and molecular dynamics simulations, we show that RNA incorporates into FUS condensates, reducing condensate density, drawing in water and ions, and enhancing local and diffusional motion. Whereas RNA binding in the dispersed phase primarily involves RGG1, within the condensed phase both LC and RGG1 contribute to RNA interactions. A diverse set of interactions between amino acids and RNA moieties, including prominent glutamine contacts, contributes to FUS‐RNA co‐condensates. RNA displaces RGG‐mediated protein contacts while enhancing LC–LC interactions, providing a molecular basis for RNA‐driven condensate interaction network remodeling. Furthermore, RNA accelerates the liquid‐to‐solid transition of FUS LC‐RGG1 condensates. Together, these results provide mechanistic insight into how RNA regulates condensate assembly, dynamics, and maturation.

Fused in sarcoma (FUS) is an RNA‐binding protein that undergoes phase separation with RNA and other cellular components, forming ribonucleoprotein (RNP) granules. While recent advances in the study of biomolecular phase separation have focused on protein–protein interactions, information on the molecular details of protein–RNA interactions within condensates remains limited. Here, we demonstrate how RNA modulates the phase separation of the low‐complexity (LC) and arginine–glycine–glycine motif (RGG1) domains of FUS—low RNA concentrations enhance protein phase separation and excess RNA disrupts it. Integrating biochemical assays, NMR spectroscopy, and molecular dynamics simulations, we show that RNA incorporates into FUS condensates, reducing condensate density, drawing in water and ions, and enhancing local and diffusional motion. Whereas RNA binding in the dispersed phase primarily involves RGG1, within the condensed phase both LC and RGG1 contribute to RNA interactions. A diverse set of interactions between amino acids and RNA moieties, including prominent glutamine contacts, contributes to FUS‐RNA co‐condensates. RNA displaces RGG‐mediated protein contacts while enhancing LC–LC interactions, providing a molecular basis for RNA‐driven condensate interaction network remodeling. Furthermore, RNA accelerates the liquid‐to‐solid transition of FUS LC‐RGG1 condensates. Together, these results provide mechanistic insight into how RNA regulates condensate assembly, dynamics, and maturation.

TAR DNA binding protein 43 (TDP-43), a key protein linked to ALS pathology, undergoes phase separation and forms functional assemblies via condensation within cells. The conserved region (CR) within its C-terminal domain (CTD) mediates self-assembly through helix–helix interactions, while the flanking intrinsically disordered regions (IDRs) contribute to phase separation through transient interactions involving aromatic and hydrophobic residues. The CTD contains ten methionine residues distributed equally between these regions, making it particularly susceptible to oxidative modifications. While methionine oxidation is known to impair TDP-43 phase separation, neither the precise mechanism nor the specific contribution of methionines in the CR compared to the IDRs has been determined. Here, we combine NMR spectroscopy and molecular dynamics (MD) simulations to reveal if and how methionine oxidation in each region differentially affects CTD structure and phase separation. To assess the change of secondary structure caused by oxidation, we measured NMR random coil chemical shift values for methionine sulfoxide. Oxidation of CR methionines disrupts helical structure and directly impairs intermolecular helical association, while oxidation of IDR methionines disrupts long-range contacts. Hence, oxidation of methionines in both regions contributes to impaired phase separation, albeit through different mechanisms. These findings establish methionines as critical redox-sensitive modulators in TDP-43 phase behavior and provide molecular insights into how oxidative stress may contribute to TDP-43 dysregulation in neurodegenerative diseases.

Aberrant aggregation of the prion-like RNA binding protein TDP-43 drives several fatal neurodegenerative proteinopathies, including amyotrophic lateral sclerosis (ALS). In this work, we define how short, specific RNAs solubilize TDP-43. These short RNAs engage and stabilize the TDP-43 RNA recognition motifs, which allosterically destabilizes a conserved helical region in the prion-like domain, thereby promoting aggregation-resistant conformers. Sequence-space mining identified short RNA chaperones with enhanced activity against TDP-43 and disease-linked variants. Enhanced short RNA chaperones mitigated aberrant TDP-43 phenotypes in optogenetic models and in ALS patient–derived and control motor neurons. In mice with cytoplasmic TDP-43 aggregation and motor neuron loss, an enhanced short RNA chaperone reduced pathological aggregation, restored TDP-43 function, and conferred neuroprotection. These results define a mechanistic and therapeutic framework for RNA-based strategies to counter TDP-43 proteinopathies.

The RNA-binding protein Fused in Sarcoma (FUS) undergoes phase separation associated with RNA processing. However, the prion-like low complexity (LC) domain of FUS forms solid-like aggregates in neurodegenerative diseases. Whether the formation of β-sheet structure associated with pathology is also physiologically/functionally relevant is debated. Similarly, if mislocalization alone or concomitant aggregation is responsible for FUS gain-of-function toxicity remains to be probed. Here, we introduce β-sheet breaking proline residues into FUS LC with the goal of preventing cross-β-driven aggregation without disrupting essential functions and phase separation. β-sheet-deficient FUS variants maintain native-like global motions, disorder, and phase separation, but no longer show a liquid-to-solid transition (LST). Biochemical partitioning, cellular localization, and auto- and cross-regulatory functions of FUS all remain essentially unchanged. Conversely, FUS-induced neurodegeneration in several Drosophila models is drastically reduced. These findings suggest a strategy for mitigating disease-related toxicity through backbone structure modulation to prevent prion-like domain protein aggregation. GRAPHICAL SUMMARY: The RNA-binding protein Fused in Sarcoma (FUS) undergoes phase separation as part of its physiological function but can aberrantly aggregate into solid-like assemblies in amyotrophic lateral sclerosis and frontotemporal dementia. To dissect the role of β-sheets in both function and pathological transition, we engineered β-sheet-preventing FUS variants via targeted proline residue insertions in the prion-like disordered region. These variants retained native structure, motions, and phase behavior yet showed dramatically reduced aggregation, both as an isolated prion-like domain and in full-length FUS. Crucially, these variants maintained a panel of FUS cellular functions that depend on FUS condensation but prevented FUS toxicity in fly models of neurodegeneration. Our findings implicate β-sheets as key drivers of FUS condensate maturation and neuronal toxicity, highlighting β-sheet modulation as a therapeutic strategy against FUS-related neurodegeneration. HIGHLIGHTS: Targeted proline additions disrupt β-sheet formation in FUS without altering native conformations, dynamics, or phase separation behavior β-sheet-deficient FUS variants prevent aggregation and liquid-to-solid transitions while retaining key biological functions In vivo models reveal attenuated toxicity of β-sheet-deficient FUS in Drosophila β-sheets are identified as central drivers of condensate maturation and neuronal death, offering a therapeutic entry point for modulating prion-like domain pathology

The DNA damage response relies on the rapid assembly of repair factors into foci with properties of liquid-liquid phase separation, driven by de novo transcription of damage-induced RNAs. 53BP1 is a key component of these condensates, yet the molecular determinants driving this process remain unknown. Here, through computational, structural and in vitro approaches, we identify the oligomerization domain of 53BP1 and its glycine-arginine-rich (GAR) motif as crucial for RNA interactions and phase separation. Biophysical characterization reveals that 53BP1-RNA condensates can progressively mature into a more stable state, and that GAR mutants display aberrant material properties. Using a cellular model of telomere fusion events, we demonstrate that the GAR motif is essential for 53BP1-mediated DNA repair, which depends on the combined contributions of RNA binding and appropriate condensate biophysical properties. Therefore, RNA-driven 53BP1 condensation is functionally required to maintain genome integrity.

Though the structural details of how RNA interacts with folded RNA-binding domains are well established, how intrinsically disordered regions (IDRs) found in a large fraction of RNA-binding proteins mediate contacts with RNA and if they contribute to binding specificity has not been extensively characterized. The human RNA-binding protein TDP-43 is associated with many RNA processing functions that require its predominantly disordered C-terminal domain (CTD) that forms disease-associated inclusions in ALS, and other neurodegenerative conditions. Here, we demonstrate that TDP-43 CTD directly interacts with RNA primarily via a region of the IDR composed of clustered positively charged residues. Large RNAs act as a multivalent scaffold for CTD monomers, inducing the α-helical segment of TDP-43 CTD to form multimeric protein-protein structures. Additionally, we probe the nucleotide base and amino acid specificity of CTD-RNA interactions, showing that arginine, aromatic and polar residues display a preference for U and G nucleic acid bases over C and A. Finally, we probe the molecular basis for the strong binding interaction between TDP-43 and G4 quadruplex structures and discover similarly avid interactions with cytosine-rich DNA I-motifs. This work deepens our understanding of how disordered regions of proteins contribute to RNA recognition, drive function, and contribute to disease.

TAR DNA binding protein 43 (TDP-43), a key protein linked to ALS pathology, undergoes phase separation and forms functional assemblies via condensation within cells. The conserved region (CR) within its C-terminal domain (CTD) mediates self-assembly through helix-helix interactions, while the flanking intrinsically disordered regions (IDRs) contribute to phase separation through transient interactions involving aromatic and hydrophobic residues. The CTD contains ten methionine residues distributed equally between these regions, making it particularly susceptible to oxidative modifications. While methionine oxidation is known to impair phase separation, neither the precise mechanism nor the specific contribution of methionines in the CR compared to the IDRs has been determined. Here, we combine NMR spectroscopy and all-atom molecular dynamics (MD) simulations to reveal if and how methionine oxidation in each region differentially affects CTD structure and phase separation. We demonstrate that all methionine residues are vulnerable to oxidation, leading to distinct regional effects: oxidation of CR methionines disrupts helical structure and directly impairs intermolecular helical association, while oxidation of IDR methionines disrupts long-range contacts. Hence, oxidation of methionines in both regions contributes to impaired phase separation, albeit through different mechanisms. These findings establish methionines as critical redox-sensitive modulators of TDP-43 phase behavior and provide molecular insights into how oxidative stress may contribute to TDP-43 dysregulation in neurodegenerative diseases.