Abstract
Liquid-liquid phase separation of proteins and nucleic acids into membraneless organelles (MLOs) spatially organizes cellular components and reactions. The RNA-binding protein heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2) carries mRNA targets in MLOs called transport granules in neurons and oligodendrocytes. At sites of local translation, hnRNPA2 is phosphorylated by the tyrosine protein kinase Fyn, releasing the mRNA for translation. Fyn recognizes targets through its SH3 domain (Fyn-SH3). However, hnRNPA2 lacks canonical SH3-binding sequences, raising the question of how Fyn-SH3 binds hnRNPA2 in phase-separated transport granules. Here, we characterize the structural details of the interaction of the hnRNPA2 low-complexity domain (LC) with Fyn-SH3 and the effect of Fyn-SH3 on hnRNPA2 phase separation. We combined in vitro microscopy and solution NMR spectroscopy to evaluate assembly of hnRNPA2 and Fyn-SH3 into in vitro phase-separated granules and probe the structural details of their interaction. We observed that Fyn-SH3 induces hnRNPA2 LC phase separation and that Fyn-SH3 is incorporated into in vitro hnRNPA2 LC granules. Moreover, we identified hnRNPA2 LC interaction sites on the surface of Fyn-SH3. Our data offer a structural view of how hnRNPA2 LC may interact with Fyn. To our knowledge, our study provides the first example of a single globular domain inducing phase separation of a disordered MLO scaffold protein.
Highlights
Liquid–liquid phase separation of proteins and nucleic acids into membraneless organelles (MLOs) spatially organizes cellular components and reactions
HnRNPA2 low-complexity domain (LC) alone does not phase-separate at these solution conditions [12]; these data suggest that FynSH3 induces phase separation of heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2) LC
We saw significant chemical shift perturbations of the residues in the 4 strand and the RT loop that are conserved across many SH3 domains [25], suggesting that these residues are important for SH3 function
Summary
We performed titration experiments using NMR to determine which regions of hnRNPA2 LC mediate interactions with Fyn-SH3. In 0 mM NaCl buffer, DIC microscopy images show that the addition of Fyn-SH3 induces the formation of hnRNPA2 LC WT liquid droplets at a concentration and solution conditions where hnRNPA2 LC alone does not phase-separate (Fig. 2D, second column, bottom panel) [12]. As the concentration of the hnRNPA2 subpeptide was increased, we observed small but increasing chemical shift perturbations of Fyn-SH3 resonances (Fig. 4B and Fig. S3A), consistent with our microscopy experiments that suggest an interaction between Fyn-SH3 and hnRNPA2 LC. The hnRNPA2 190 –303 phaseseparated upon the addition of equimolar Fyn-SH3, so we were unable to observe any significant chemical shift perturbations (Fig. S3, C–E) It appears that protein–protein interaction occurs, but LLPS precludes straightforward evaluation of the site-specific details of the interaction by NMR. This subpeptide does have more proline residues (7 total) than the 266 – 341 subpeptide; this region may be a major contributor to Fyn-SH3 interaction with hnRNPA2
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