RNA promotes liquid-liquid phase separation (LLPS) to build membrane-less compartments in cells. How distinct molecular compositions are established and maintained in these liquid compartments is unknown. Here we report that secondary structure allows mRNAs to self-associate and determines if 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.

Highlights d FUS multimer interacts dynamically with RNA, resulting in liquid-like condensates d ALS/FTD-linked FUS mutations in arginine lead to defective RNA binding d ALS/FTD-linked FUS mutations in glycine induce quick loss of condensate fluidity d Karyopherin-b2 reverses mutant defects and recovers wildtype FUS properties

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.

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.

MicroRNA-mediated gene silencing is a fundamental mechanism in the regulation of gene expression. It remains unclear how the efficiency of RNA silencing could be influenced by RNA-binding proteins associated with the microRNA-induced silencing complex (miRISC). Here we report that fused in sarcoma (FUS), an RNA-binding protein linked to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), interacts with the core miRISC component AGO2 and is required for optimal microRNA-mediated gene silencing. FUS promotes gene silencing by binding to microRNA and mRNA targets, as illustrated by its action on miR-200c and its target ZEB1. A truncated mutant form of FUS that leads its carriers to an aggressive form of ALS, R495X, impairs microRNA-mediated gene silencing. The C. elegans homolog fust-1 also shares a conserved role in regulating the microRNA pathway. Collectively, our results suggest a role for FUS in regulating the activity of microRNA-mediated silencing.

Abstract:Many subcellular structures assemble via liquid-liquid phase separation (LLPS) to form compartments without membranes. Though it has been shown that RNA is a central driver and modulator of LLPS, it is not yet known how these liquid droplets establish and maintain individual identities. Here we examine how mRNAs are recruited to or excluded from liquid compartments based on their sequence and ability to self-associate. We find that the specific secondary structure of a cyclin mRNA is required for it to assemble into distinct droplets and be excluded from other droplets containing functionally-unrelated mRNAs. This molecular mechanism explains how sequence-encoded shape information in RNA promotes the coexistence of the diverse array of RNA-rich liquid compartments found in a single cell.peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/233817 doi: bioRxiv preprint first posted online Dec. 13, 2017; 2 One Sentence Summary:Identity in cellular, phase-separated compartments emerges from RNA-RNA complexes encoded by mRNA secondary structures. Main Text:The formation of non-membrane bound organelles through the condensation of macromolecules is a newly appreciated mechanism of intracellular organization. These condensates often display liquid-like properties and form through liquid-liquid phase separation (LLPS) (1, 2). The growing list of LLPS-assembled compartments includes the nucleolus, RNA granules, cell signaling hubs, the spindle matrix, chromatin, the synaptonemal complex, and many pathological neuronal granules (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Despite the growing appreciation of the variety of liquid-like assemblies employed in diverse cellular processes, a fundamental unsolved problem is how liquid droplets recruit distinct constituents and retain independent identities, rather than fusing into a singular compartment. This is especially remarkable given the fact that many of the constituents are highly disordered proteins and the droplets they form display such a propensity to fuse (2). RNA has been shown to be a driver of LLPS and can modulate the material properties of droplets (13-20), yet there is little known about how RNA can impact the identity and maintenance of coexisting liquid compartments. Here we show that mRNA secondary structure is required for droplet identity and likely acts through higher-order interactions between mRNAs and RNA-binding proteins. This illustrates how molecular scale interactions can encode the identity and emergent properties of micron-scale liquid compartments in cells.peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/233817 doi: bioRxiv preprint first posted online Dec. 13, 2017; 3 Whi3 is a polyQ-containing RNA-binding protein identified in Saccharomyces cerevisiae through its role in cell size control (21...

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