Condensate formation of the human RNA-binding protein SMAUG1 is controlled by its intrinsically disordered regions and interactions with 14-3-3 proteins

Abstract

SMAUG1 is a human RNA-binding protein that is known to be dysregulated in a wide range of diseases. It is evolutionarily conserved and has been shown to form condensates containing translationally repressed RNAs. This indicates that condensation is central to proper SMAUG1 function; however, the factors governing condensation are largely unknown. In this work, we show that SMAUG1 drives the formation of liquid-like condensates in cells through its non-conventional C-terminal prion-like disordered region. We use biochemical assays to show that this liquid-liquid phase separation is independent of RNA binding and does not depend on other large, disordered regions that potentially harbor several binding sites for partner proteins. Using a combination of computational predictions, structural modeling, in vitro and in cell measurements, we also show that SMAUG1-driven condensation is negatively regulated by direct interactions with the members of the 14-3-3 protein family. These interactions are mediated by four distinct phospho-regulated short linear motifs embedded in the disordered regions of SMAUG1, working synergistically. Interactions between SMAUG1 and 14-3-3 proteins drive the dissolution of condensates, alter the dynamics of the condensed state, and are likely to be intertwined with currently unknown regulatory mechanisms. Our results provide information on how SMAUG1 phase separation is regulated and the first known instance of 14-3-3 proteins being able to completely dissolve condensates by directly interacting with a phase separation driver, which might be a general mechanism in cells to regulate biological condensation.

14-3-3 binding regulates Tau assembly and microtubule association

“…Stoichiometric binding pocket interactions of 14-3-3ζ dimers with Tau monomers phosphorylated at S214 and S324 make Tau molecules unavailable for multivalent interactions needed for LLPS and, therefore, counteract their co-condensation. The suppression of biomolecular condensation by 14-3-3 binding has been suggested for other physiological and disease-associated 14-3-3 binding partners 49,50 , however, the molecular mechanisms behind this process previously remained uncertain.…”

Structural basis for binding of Drosophila Smaug to the GPCR Smoothened and to the germline inducer Oskar

“…Often the recognition of target proteins by F-box proteins, such as Slmb, depends on prior phosphorylation of the target ( 66 – 69 ). Mouse and human SAMD4A/Smaug1 were reported to interact with 14-3-3 proteins ( 18 , 70 ), a protein family known to recognize phosphorylated sequences within their protein partners ( 71 ). We detected the Drosophila Smaug interaction to 14-3-3ζ and 14-3-3ε in our ReLo assays ( SI Appendix , Fig.…”

Structural basis for binding of Smaug to the GPCR Smoothened and to the germline inducer Oskar

“…Often the recognition of target proteins by F-box proteins, such as Slmb, depends on prior phosphorylation of the target (Morais-de-Sá et al , 2013; Mason & Laman, 2020; Frescas & Pagano, 2008; Jia et al , 2005). Mouse and human SAMD4A/Smaug1 were reported to interact with 14-3-3 proteins (Chen et al , 2014b; Fehilly et al , 2023), a protein family known to recognize phosphorylated sequences within their protein partners (Obsilova & Obsil, 2022). We detected the Drosophila Smaug interaction to 14-3-3ζ and 14-3-3ε in our ReLo assays ( Supplemental Figure 8C ), suggesting that Smaug is phosphorylated in these cells.…”