Liquid‐Liquid Phase Separation of hnRNP A2 and Implications for Structure and Disease Propagation

A family of RNA‐binding proteins characterized by the presence of putatively disordered domains containing high glutamine, asparagine, and aromatic content have recently been linked to the formation of intracellular puncta and neurodegenerative disease‐associated aggregates. One member of this family, heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2), is known to aggregate and contribute to the pathogenesis of multisystem proteinopathy (MSP) and inclusion body myositis (IBM). HnRNPs dynamically associate into large protein‐RNA complexes, termed ribonucleoprotein (RNP) granules, which have important roles in RNA processing, transport, and sequestration. These granules may act as nucleation sites for aggregates in neurodegenerative diseases. The precise mechanism by which hnRNPA2 exerts this toxic influence is unknown, therefore we performed structural studies on hnRNP A2 to determine how granules nucleate aggregates. Here, we used differential interference contrast microscopy and fluorescence recovery after photo bleaching to show that the isolated disordered domain of hnRNPA2 and its disease mutant D290V form a liquid‐liquid phase separated state mimicking RNP granules in vitro. We used the same methods to investigate how the full length protein changed the dynamics of the liquid‐liquid phase separation. Furthermore, using solution NMR, we determined the structure of monomeric wild‐type and D290V hnRNPA2. These results lay the foundation for observing liquid‐liquid phase separated states of disorder proteins with atomistic resolution.