Interplay of folded domains and the disordered low-complexity domain in mediating hnRNPA1 phase separation

Liquid-liquid phase separation has emerged as a new paradigm for the compartmentalization of cells without membranes. Intrinsically disordered low-complexity domains (LCDs) are often sufficient for mediating phase separation, but how their phase behavior is modulated by the presence of attached folded domains is incompletely understood. Here, we interrogate the interplay between folded and disordered domains of the RNA-binding protein hnRNPA1, which localizes to stress granules under conditions of stress. The LCD of hnRNPA1 is sufficient for mediating phase separation in vitro. However, the stimulus-responsive phase behavior of a number of domain deletion constructs suggests that the folded RRM domains contribute to phase separation, even in the absence of RNA. Small-angle X-ray scattering experiments show that hnRNPA1 adopts compact conformations at low ionic strength, that the protein expands considerably with increasing ionic strength, and that it reaches a maximal plateau at 300 mM NaCl. These data point to electrostatically mediated interactions that compact hnRNPA1. NMR experiments and coarse-grained MD simulations showed that the LCD interacts transiently with the RRMs, providing a mechanistic explanation for the observed stimulus-responsiveness of phase separation. Disordered and folded domains that have evolved together likely encode a multitude of interactions that can modify and regulate phase separation.