Biology subjects

Sojitra, K. A.

Publications and source records attributed to Sojitra, K. A..

3 recordsLinked to original sources

bioRxiv2025

Molecular Basis of EWS Interdomain Self-Association and Its Role in Condensate Formation

Ewing sarcoma, the second most common pediatric bone and soft tissue cancer, is caused by aberrant fusion of the EWS low-complexity domain (EWSLCD) to the DNA-binding domain of the transcription factor FLI1. The resulting fusion, EWS::FLI1, directly interacts with and engages in a dynamic interplay with EWS that drives tumorigenesis and regulates the function of both proteins. While EWSLCD is known to promote self-association, the role of the RNA-binding domains (RBDs) of EWS, which include RGG repeat regions and a structured RNA-recognition motif (RRM), remains less well understood. Here, we investigate the interplay between EWSLCD and RBDs using biomolecular condensation assays, microscopy, NMR spectroscopy, and molecular simulations. Our studies reveal that RBDs differentially influence EWSLCD condensate formation and suggest that electrostatics and polypeptide-chain length likely contribute to this interaction. NMR spectroscopy and molecular dynamics simulations further demonstrate that EWSLCD and the central RNA-binding region, comprising the RRM and RGG2 domains, engage in transient, non-specific interactions that are broadly distributed across both regions and involve diverse residue types. Specifically, tyrosine, polar residues, and proline within EWSLCD preferentially interact with arginine, glycine, and proline residues in the RBD. Atomistic simulations of EWS confirm that the full-length protein exhibits a similar interaction profile with conserved chemical specificity, supporting a model in which a network of weak, distributed interdomain contacts underlies EWS self-association. Together, these findings provide molecular insight into the mechanisms of EWS condensate formation and lay the groundwork for understanding how interdomain interactions regulate EWS and EWS::FLI1 function. SIGNIFICANCEThis study shows that the RNA-binding regions of the RNA-binding protein EWS subtly tune biomolecular condensation through numerous transient contacts with the low complexity domain that is common to both EWS and the oncogenic fusion EWS::FLI1. These results provide insight into condensate formation of EWS, which may be useful for understanding the oncogenic mechanisms of EWS::FLI1 and broadly, into pathogenic biomolecular condensate formation.

biophysics↗

bioRxiv2023

Insights into Molecular Diversity within the FET Family: Unraveling Phase Separation of the N-Terminal Low Complexity Domain from RNA-Binding Protein EWS

The FET family proteins, which includes FUS, EWS, and TAF15, are RNA chaperones instrumental in processes such as mRNA maturation, transcriptional regulation, and the DNA damage response. These proteins have clinical significance: chromosomal rearrangements in FET proteins are implicated in Ewing family tumors and related sarcomas. Furthermore, point mutations in FUS and TAF15 are associated with neurodegenerative conditions like amyotrophic lateral sclerosis and frontotemporal lobar dementia. The fusion protein EWS::FLI1, the causative mutation of Ewing sarcoma, arises from a genomic translocation that fuses the low-complexity domain (LCD) of EWS (EWSLCD) with the DNA binding domain of the ETS transcription factor FLI1. This fusion not only alters transcriptional programs but also hinders native EWS functions like splicing. However, the precise function of the intrinsically disordered EWSLCD is still a topic of active investigation. Due to its flexible nature, EWSLCD can form transient interactions with itself and other biomolecules, leading to the formation of biomolecular condensates through phase separation - a mechanism thought to be central to the oncogenicity of EWS::FLI1. In our study, we used paramagnetic relaxation enhancement NMR, analytical ultracentrifugation, light microscopy, and all-atom molecular dynamics (MD) simulations to better understand the self-association and phase separation tendencies of EWSLCD. Our aim was to elucidate the molecular events that underpin EWSLCD-mediated biomolecular condensation. Our NMR data suggest tyrosine residues primarily drive the interactions vital for EWSLCD phase separation. Moreover, a higher density and proximity of tyrosine residues amplify the likelihood of condensate formation. Atomistic MD simulations and hydrodynamic experiments revealed that the tyrosine-rich N and C-termini tend to populate compact conformations, establishing unique contact networks, that are connected by a predominantly extended, tyrosine-depleted, linker region. MD simulations provide critical input on the relationship between contacts formed within a single molecule (intramolecular) and inside the condensed phase (intermolecular), and changes in protein conformations upon condensation. These results offer deeper insights into the condensate-forming abilities of the FET proteins and highlights unique structural and functional nuances between EWS and its counterparts, FUS and TAF15.

biophysics↗

bioRxiv2023

Determinants of Disordered Protein Co-Assembly Into Discrete Condensed Phases

Cells harbor numerous mesoscale membraneless compartments that house specific biochemical processes and perform distinct cellular functions. These protein and RNA-rich bodies are thought to form through multivalent interactions among proteins and nucleic acids resulting in demixing via liquid-liquid phase separation (LLPS). Proteins harboring intrinsically disordered regions (IDRs) predominate in membraneless organelles. However, it is not known whether IDR sequence alone can dictate the formation of distinct condensed phases. We identified a pair of IDRs capable of forming spatially distinct condensates when expressed in cells. When reconstituted in vitro, these model proteins do not co-partition, suggesting condensation specificity is encoded directly in the polypeptide sequences. Through computational modeling and mutagenesis, we identified the amino acids and chain properties governing homotypic and heterotypic interactions that direct selective condensation. These results form the basis of physicochemical principles that may direct subcellular organization of IDRs into specific condensates and reveal an IDR code that can guide construction of orthogonal membraneless compartments.

biophysics↗