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RNA-driven phase separation is emerging as a promising approach for engineering biomolecular condensates with diverse functionalities. Condensates form thanks to weak yet specific RNA–RNA interactions established by design via complementary sequence domains. Here, we demonstrate how RNA condensates formed by star-shaped RNA motifs, or nanostars, can be dynamically controlled when the motifs include additional linear or branch-loop domains that facilitate access of regulatory RNA molecules to the nanostar interaction domains. We show that condensates dissolve in the presence of RNA “invaders” that occlude selected nanostar bonds and reduce the valency of the nanostars, preventing phase separation. We further demonstrate that the introduction of “anti-invader” strands, complementary to the invaders, makes it possible to restore condensate formation. An important aspect of our experiments is that we demonstrate these behaviors in one-pot reactions, where RNA nanostars, invaders, and anti-invaders are simultaneously transcribed in vitro using short DNA templates. Our results lay the groundwork for engineering RNA-based assemblies with tunable, reversible condensation, providing a promising toolkit for synthetic biology applications requiring responsive, self-organizing biomolecular materials.
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Membrane‐dependent assembly of Bruton's tyrosine kinase mediated by the Proline‐rich region and SH3 domain
Abstract Cellular membranes provide a unique platform for interactions that drive emergent behaviors in protein dynamics and cellular signaling, distinct from those observed in solution. We investigated the proline‐rich region (PRR) and Src Homology 3 (SH3) domains of Bruton's tyrosine kinase (Btk) and its phase separation driven by the weak interactions of regulatory domains at membrane surfaces. Using supported lipid bilayers (SLBs) and giant unilamellar vesicles (GUVs), we demonstrate that membrane localization amplifies weak PRR‐SH3 interactions, enabling the formation of higher‐order assemblies and phase‐separated condensates. These assemblies, previously undescribed by solution‐state studies, are supported by reductions in the lateral diffusion of membrane‐bound Btk molecules and the stabilization of reversible condensates at the membrane surface. Constructs containing the native PRR and SH3 domains reliably formed membrane‐associated clusters, while mutation or deletion of these domains lessened changes in diffusion and impaired condensate formation. Our findings establish the membrane as an essential mediator of PRR‐SH3‐driven phase separation in Btk, thereby advancing our understanding of membrane‐specific regulation in signaling protein dynamics.
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- Award ID(s):
- 2238109
- PAR ID:
- 10656832
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Protein Science
- Volume:
- 34
- Issue:
- 8
- ISSN:
- 0961-8368
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1002/pro.70213
