Ubiquilin 2 modulates ALS/FTD-linked FUS-RNA complex dynamics and stress granule formation.

The ubiquitin-like protein ubiquilin 2 (UBQLN2) has been genetically and pathologically linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but its normal cellular functions are not well understood. In a search for UBQLN2-interacting proteins, we found an enrichment of stress granule (SG) components, including ALS/FTD-linked heterogeneous ribonucleoprotein fused in sarcoma (FUS). Through the use of an optimized SG detection method, we observed UBQLN2 and its interactors at SGs. A low complexity, Sti1-like repeat region in UBQLN2 was sufficient for its localization to SGs. Functionally, UBQLN2 negatively regulated SG formation. UBQLN2 increased the dynamics of FUS-RNA interaction and promoted the fluidity of FUS-RNA complexes at a single-molecule level. This solubilizing effect corresponded to a dispersal of FUS liquid droplets in vitro and a suppression of FUS SG formation in cells. ALS-linked mutations in UBQLN2 reduced its association with FUS and impaired its function in regulating FUS-RNA complex dynamics and SG formation. These results reveal a previously unrecognized role for UBQLN2 in regulating the early stages of liquid-liquid phase separation by directly modulating the fluidity of protein-RNA complexes and the dynamics of SG formation.

RNA promotes phase separation of glycolysis enzymes into yeast G bodies in hypoxia.

In hypoxic stress conditions, glycolysis enzymes assemble into singular cytoplasmic granules called glycolytic (G) bodies. G body formation in yeast correlates with increased glucose consumption and cell survival. However, the physical properties and organizing principles that define G body formation are unclear. We demonstrate that glycolysis enzymes are non-canonical RNA binding proteins, sharing many common mRNA substrates that are also integral constituents of G bodies. Targeting nonspecific endoribonucleases to G bodies reveals that RNA nucleates G body formation and maintains its structural integrity. Consistent with a phase separation mechanism of biogenesis, recruitment of glycolysis enzymes to G bodies relies on multivalent homotypic and heterotypic interactions. Furthermore, G bodies fuse in vivo and are largely insensitive to 1,6-hexanediol, consistent with a hydrogel-like composition. Taken together, our results elucidate the biophysical nature of G bodies and demonstrate that RNA nucleates phase separation of the glycolysis machinery in response to hypoxic stress.

mRNA structure determines specificity of a polyQ-driven phase separation.

RNA promotes liquid-liquid phase separation (LLPS) to build membraneless compartments in cells. How distinct molecular compositions are established and maintained in these liquid compartments is unknown. Here, we report that secondary structure allows messenger RNAs (mRNAs) to self-associate and determines whether an mRNA is recruited to or excluded from liquid compartments. The polyQ-protein Whi3 induces conformational changes in RNA structure and generates distinct molecular fluctuations depending on the RNA sequence. These data support a model in which structure-based, RNA-RNA interactions promote assembly of distinct droplets and protein-driven, conformational dynamics of the RNA maintain this identity. Thus, the shape of RNA can promote the formation and coexistence of the diverse array of RNA-rich liquid compartments found in a single cell.