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.

Model organisms are widely used in research as accessible and convenient systems to study a particular area or question in biology. Traditionally only a handful of organisms have been widely studied, but modern research tools are enabling researchers to extend the set of model organisms to include less-studied and more unusual systems. This Forum highlights a range of 'non-model model organisms' as emerging systems for tackling questions across the whole spectrum of biology (and beyond), the opportunities and challenges, and the outlook for the future.

Biomolecular condensates organize biochemistry, yet little is known about how cells control the position and scale of these structures. In cells, condensates often appear as relatively small assemblies that do not coarsen into a single droplet despite their propensity to fuse. Here we report that ribonucleoprotein condensates of the Q-rich protein Whi3 interact with the endoplasmic reticulum, prompting us to examine how membrane association controls condensate size. Reconstitution reveals that membrane recruitment promotes Whi3 condensation under physiological conditions. These assemblies rapidly arrest, resembling size distributions seen in cells. The temporal ordering of molecular interactions and the slow diffusion of membrane-bound complexes can limit condensate size. Our experiments reveal a tradeoff between locally-enhanced protein concentration at membranes, favoring condensation, and an accompanying reduction in diffusion, restricting coarsening. Given that many condensates bind endomembranes, we predict that the biophysical properties of lipid bilayers are key for controlling condensate sizes throughout the cell.

Fadero et al. present lateral interference tilted excitation (LITE) microscopy–a tilted light-sheet method to illuminate high-numerical-aperture objectives for fluorescence microscopy. LITE can be implemented unobtrusively on most microscope systems and combines low photodamage with high resolution and efficient detection in imaging fluorescent organisms.

Biomolecular condensation is a way of organizing cytosol in which proteins and nucleic acids coassemble into compartments. In the multinucleate filamentous fungus Ashbya gossypii, the RNA-binding protein Whi3 regulates the cell cycle and cell polarity through forming macromolecular structures that behave like condensates. Whi3 has distinct spatial localizations and mRNA targets, making it a powerful model for how, when, and where specific identities are established for condensates. We identified residues on Whi3 that are differentially phosphorylated under specific conditions and generated mutants that ablate this regulation. This yielded separation of function alleles that were functional for either cell polarity or nuclear cycling but not both. This study shows that phosphorylation of individual residues on molecules in biomolecular condensates can provide specificity that gives rise to distinct functional identities in the same cell.

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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