mRNA structure determines specificity of a polyQ-driven phase separation

Abstract

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...

Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures

“…To this end, we tested all mutations in the 1-1000 context with recombinant Whi3 protein. Whi3 has previously been shown to undergo sequence-specific RNA-dependent LLPS (Langdon et al, 2018;Zhang et al, 2015). We observed no obvious difference between any of the mutant RNAs and the wildtype 1-1000nt sequence with condensing Whi3 protein (Fig.…”

“…All experiments were performed with His-tagged N-protein. Whi3 was purified according to our established protocols (Langdon et al, 2018;Zhang et al, 2015).…”

“…Wildtype +dsRNAb +dsRNAd +ssRNAa -ssRNA Restore pairing In vitro transcription: was carried out according to our established protocols (Langdon et al, 2018). Orf1ab templates were synthesized (IDT) and cloned into pJet (ThermoFisher Scientific K1231) using blunt end cloning.…”

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Functional Implications of Intracellular Phase Transitions

“…The chemical environment inside condensates is expected to be significantly different from the bulk cytoplasm. ,, The rules that determine which proteins and nucleic acids are recruited into or are excluded from specific organelles remain poorly understood. , Here, we approach this problem from purely thermodynamic considerations. From this vantage point, equalization of chemical potentials across a phase boundary will be the main determinant of the extent to which different molecules are partitioned into or excluded from condensates.…”