RNA contributions to the form and function of biomolecular condensates.

Biomolecular condensation partitions cellular contents and has important roles in stress responses, maintaining homeostasis, development and disease. Many nuclear and cytoplasmic condensates are rich in RNA and RNA-binding proteins (RBPs), which undergo liquid-liquid phase separation (LLPS). Whereas the role of RBPs in condensates has been well studied, less attention has been paid to the contribution of RNA to LLPS. In this Review, we discuss the role of RNA in biomolecular condensation and highlight considerations for designing condensate reconstitution experiments. We focus on RNA properties such as composition, length, structure, modifications and expression level. These properties can modulate the biophysical features of native condensates, including their size, shape, viscosity, liquidity, surface tension and composition. We also discuss the role of RNA-protein condensates in development, disease and homeostasis, emphasizing how their properties and function can be determined by RNA. Finally, we discuss the multifaceted cellular functions of biomolecular condensates, including cell compartmentalization through RNA transport and localization, supporting catalytic processes, storage and inheritance of specific molecules, and buffering noise and responding to stress.

Genomic RNA Elements Drive Phase Separation of the SARS-CoV-2 Nucleocapsid.

We report that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with viral RNA. N-protein condenses with specific RNA genomic elements under physiological buffer conditions and condensation is enhanced at human body temperatures (33°C and 37°C) and reduced at room temperature (22°C). RNA sequence and structure in specific genomic regions regulate N-protein condensation while other genomic regions promote condensate dissolution, potentially preventing aggregation of the large genome. At low concentrations, N-protein preferentially crosslinks to specific regions characterized by single-stranded RNA flanked by structured elements and these features specify the location, number, and strength of N-protein binding sites (valency). Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is RNA sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules, and therefore presents a screenable process for identifying antiviral compounds effective against SARS-CoV-2.

The Control Centers of Biomolecular Phase Separation: How Membrane Surfaces, PTMs, and Active Processes Regulate Condensation.

Biomolecular condensation is emerging as an essential process for cellular compartmentalization. The formation of biomolecular condensates can be driven by liquid-liquid phase separation, which arises from weak, multivalent interactions among proteins and nucleic acids. A substantial body of recent work has revealed that diverse cellular processes rely on biomolecular condensation and that aberrant phase separation may cause disease. Many proteins display an intrinsic propensity to undergo phase separation. However, the mechanisms by which cells regulate phase separation to build functional condensates at the appropriate time and location are only beginning to be understood. Here, we review three key cellular mechanisms that enable the control of biomolecular phase separation: membrane surfaces, post-translational modifications, and active processes. We discuss how these mechanisms may function in concert to provide robust control over biomolecular condensates and suggest new research avenues that will elucidate how cells build and maintain these key centers of cellular compartmentalization.

Evolution and Physiology of Amphibious Yeasts.

Since the emergence of the first fungi some 700 million years ago, unicellular yeast-like forms have emerged multiple times in independent lineages via convergent evolution. While tens to hundreds of millions of years separate the independent evolution of these unicellular organisms, they share remarkable phenotypic and metabolic similarities, and all have streamlined genomes. Yeasts occur in every aquatic environment yet examined. Many species are aquatic; perhaps most are amphibious. How these species have evolved to thrive in aquatic habitats is fundamental to understanding functions and evolutionary mechanisms in this unique group of fungi. Here we review the state of knowledge of the physiological and ecological diversity of amphibious yeasts and their key evolutionary adaptations enabling survival in aquatic habitats. We emphasize some genera previously thought to be exclusively terrestrial. Finally, we discuss the ability of many yeasts to survive in extreme habitats and how this might lend insight into ecological plasticity, including amphibious lifestyles.

Curvature sensing as an emergent property of multiscale assembly of septins.

The ability of cells to sense and communicate their shape is central to many of their functions. Much is known about how cells generate complex shapes, yet how they sense and respond to geometric cues remains poorly understood. Septins are GTP-binding proteins that localize to sites of micrometer-scale membrane curvature. Assembly of septins is a multistep and multiscale process, but it is unknown how these discrete steps lead to curvature sensing. Here, we experimentally examine the time-dependent binding of septins at different curvatures and septin bulk concentrations. These experiments unexpectedly indicated that septins' curvature preference is not absolute but rather is sensitive to the combinations of membrane curvatures present in a reaction, suggesting that there is competition between different curvatures for septin binding. To understand the physical underpinning of this result, we developed a kinetic model that connects septins' self-assembly and curvature-sensing properties. Our experimental and modeling results are consistent with curvature-sensitive assembly being driven by cooperative associations of septin oligomers in solution with the bound septins. When combined, the work indicates that septin curvature sensing is an emergent property of the multistep, multiscale assembly of membrane-bound septins. As a result, curvature preference is not absolute and can be modulated by changing the physicochemical and geometric parameters involved in septin assembly, including bulk concentration, and the available membrane curvatures. While much geometry-sensitive assembly in biology is thought to be guided by intrinsic material properties of molecules, this is an important example of how curvature sensing can arise from multiscale assembly of polymers.

PRD-2 mediates clock-regulated perinuclear localization of clock gene RNAs within the circadian cycle of Neurospora.

The transcription-translation negative feedback loops underlying animal and fungal circadian clocks are remarkably similar in their molecular regulatory architecture and, although much is understood about their central mechanism, little is known about the spatiotemporal dynamics of the gene products involved. A common feature of these circadian oscillators is a significant temporal delay between rhythmic accumulation of clock messenger RNAs (mRNAs) encoding negative arm proteins, for example, frq in Neurospora and Per1-3 in mammals, and the appearance of the clock protein complexes assembled from the proteins they encode. Here, we report use of single-molecule RNA fluorescence in situ hybridization (smFISH) to show that the fraction of nuclei actively transcribing the clock gene frq changes in a circadian manner, and that these mRNAs cycle in abundance with fewer than five transcripts per nucleus at any time. Spatial point patterning statistics reveal that frq is spatially clustered near nuclei in a time of day-dependent manner and that clustering requires an RNA-binding protein, PRD-2 (PERIOD-2), recently shown also to bind to mRNA encoding another core clock component, casein kinase 1. An intrinsically disordered protein, PRD-2 displays behavior in vivo and in vitro consistent with participation in biomolecular condensates. These data are consistent with a role for phase-separating RNA-binding proteins in spatiotemporally organizing clock mRNAs to facilitate local translation and assembly of clock protein complexes.

Dilute phase oligomerization can oppose phase separation and modulate material properties of a ribonucleoprotein condensate.

SignificanceA large subclass of biomolecular condensates are linked to RNA regulation and are known as ribonucleoprotein (RNP) bodies. While extensive work has identified driving forces for biomolecular condensate formation, relatively little is known about forces that oppose assembly. Here, using a fungal RNP protein, Whi3, we show that a portion of its intrinsically disordered, glutamine-rich region modulates phase separation by forming transient alpha helical structures that promote the assembly of dilute phase oligomers. These oligomers detour Whi3 proteins from condensates, thereby impacting the driving forces for phase separation, the protein-to-RNA ratio in condensates, and the material properties of condensates. Our findings show how nanoscale conformational and oligomerization equilibria can influence mesoscale phase equilibria.

Design considerations for analyzing protein translation regulation by condensates.

One proposed role for biomolecular condensates that contain RNA is translation regulation. In several specific contexts, translation has been shown to be modulated by the presence of a phase-separating protein and under conditions which promote phase separation, and likely many more await discovery. A powerful tool for determining the rules for condensate-dependent translation is the use of engineered RNA sequences, which can serve as reporters for translation efficiency. This Perspective will discuss design features to consider in engineering RNA reporters to determine the role of phase separation in translational regulation. Specifically, we will cover (i) how to engineer RNA sequence to recapitulate native protein/RNA interactions, (ii) the advantages and disadvantages for commonly used reporter RNA sequences, and (iii) important control experiments to distinguish between binding- and condensation-dependent translational repression. The goal of this review is to promote the design and application of faithful translation reporters to demonstrate a physiological role of biomolecular condensates in translation.

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

Nucleocapsid protein (N-protein) is required for multiple steps in betacoronaviruses replication. SARS-CoV-2-N-protein condenses with specific viral RNAs at particular temperatures making it a powerful model for deciphering RNA sequence specificity in condensates. We identify two separate and distinct double-stranded, RNA motifs (dsRNA stickers) that promote N-protein condensation. These dsRNA stickers are separately recognized by N-protein's two RNA binding domains (RBDs). RBD1 prefers structured RNA with sequences like the transcription-regulatory sequence (TRS). RBD2 prefers long stretches of dsRNA, independent of sequence. Thus, the two N-protein RBDs interact with distinct dsRNA stickers, and these interactions impart specific droplet physical properties that could support varied viral functions. Specifically, we find that addition of dsRNA lowers the condensation temperature dependent on RBD2 interactions and tunes translational repression. In contrast RBD1 sites are sequences critical for sub-genomic (sg) RNA generation and promote gRNA compression. The density of RBD1 binding motifs in proximity to TRS-L/B sequences is associated with levels of sub-genomic RNA generation. The switch to packaging is likely mediated by RBD1 interactions which generate particles that recapitulate the packaging unit of the virion. Thus, SARS-CoV-2 can achieve biochemical complexity, performing multiple functions in the same cytoplasm, with minimal protein components based on utilizing multiple distinct RNA motifs that control N-protein interactions.

A New Lens for RNA Localization: Liquid-Liquid Phase Separation.

RNA localization mechanisms have been intensively studied and include localized protection of mRNA from degradation, diffusion-coupled local entrapment of mRNA, and directed transport of mRNAs along the cytoskeleton. While it is well understood how cells utilize these three mechanisms to organize mRNAs within the cytoplasm, a newly appreciated mechanism of RNA localization has emerged in recent years in which mRNAs phase-separate and form liquid-like droplets. mRNAs both contribute to condensation of proteins into liquid-like structures and are themselves regulated by being incorporated into membraneless organelles. This ability to condense into droplets is in many instances contributing to previously appreciated mRNA localization phenomena. Here we review how phase separation enables mRNAs to selectively and efficiently colocalize and be coregulated, allowing control of gene expression in time and space.

RNA Controls PolyQ Protein Phase Transitions.

Compartmentalization in cells is central to the spatial and temporal control of biochemistry. In addition to membrane-bound organelles, membrane-less compartments form partitions in cells. Increasing evidence suggests that these compartments assemble through liquid-liquid phase separation. However, the spatiotemporal control of their assembly, and how they maintain distinct functional and physical identities, is poorly understood. We have previously shown an RNA-binding protein with a polyQ-expansion called Whi3 is essential for the spatial patterning of cyclin and formin transcripts in cytosol. Here, we show that specific mRNAs that are known physiological targets of Whi3 drive phase separation. mRNA can alter the viscosity of droplets, their propensity to fuse, and the exchange rates of components with bulk solution. Different mRNAs impart distinct biophysical properties of droplets, indicating mRNA can bring individuality to assemblies. Our findings suggest that mRNAs can encode not only genetic information but also the biophysical properties of phase-separated compartments.

The Unsolved Problem of How Cells Sense Micron-Scale Curvature.

Membrane curvature is a fundamental feature of cells and their organelles. Much of what we know about how cells sense curved surfaces comes from studies examining nanometer-sized molecules on nanometer-scale curvatures. We are only just beginning to understand how cells recognize curved topologies at the micron scale. In this review, we provide the reader with an overview of our current understanding of how cells sense and respond to micron-scale membrane curvature.

Role of spatial patterning of N-protein interactions in SARS-CoV-2 genome packaging.

Viruses must efficiently and specifically package their genomes while excluding cellular nucleic acids and viral subgenomic fragments. Some viruses use specific packaging signals, which are conserved sequence or structure motifs present only in the full-length genome. Recent work has shown that viral proteins important for packaging can undergo liquid-liquid phase separation (LLPS), in which one or two viral nucleic acid binding proteins condense with the genome. The compositional simplicity of viral components lends itself well to theoretical modeling compared with more complex cellular organelles. Viral LLPS can be limited to one or two viral proteins and a single genome that is enriched in LLPS-promoting features. In our previous study, we observed that LLPS-promoting sequences of severe acute respiratory syndrome coronavirus 2 are located at the 5' and 3' ends of the genome, whereas the middle of the genome is predicted to consist mostly of solubilizing elements. Is this arrangement sufficient to drive single genome packaging, genome compaction, and genome cyclization? We addressed these questions using a coarse-grained polymer model, LASSI, to study the LLPS of nucleocapsid protein with RNA sequences that either promote LLPS or solubilization. With respect to genome cyclization, we find the most optimal arrangement restricts LLPS-promoting elements to the 5' and 3' ends of the genome, consistent with the native spatial patterning. Genome compaction is enhanced by clustered LLPS-promoting binding sites, whereas single genome packaging is most efficient when binding sites are distributed throughout the genome. These results suggest that many and variably positioned LLPS-promoting signals can support packaging in the absence of a singular packaging signal which argues against necessity of such a feature. We hypothesize that this model should be generalizable to multiple viruses as well as cellular organelles such as paraspeckles, which enrich specific long RNA sequences in a defined arrangement.

Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa.

Single molecule RNA-FISH (smFISH) is a valuable tool for analysis of mRNA spatial patterning in fixed cells that is underutilized in filamentous fungi. A primary complication for fixed-cell imaging in filamentous fungi is the need for enzymatic cell wall permeabilization, which is compounded by considerable variability in cell wall composition between species. smFISH adds another layer of complexity due to a requirement for RNase free conditions. Here, we describe the cloning, expression, and purification of a chitinase suitable for supplementation of a commercially available RNase-free enzyme preparation for efficient permeabilization of the Neurospora cell wall. We further provide a method for smFISH in Neurospora which includes a tool for generating numerical data from images that can be used in downstream customized analysis protocols.

Septins and Generation of Asymmetries in Fungal Cells.

Polarized growth is critical for the development and maintenance of diverse organisms and tissues but particularly so in fungi, where nutrient uptake, communication, and reproduction all rely on cell asymmetries. To achieve polarized growth, fungi spatially organize both their cytosol and cortical membranes. Septins, a family of GTP-binding proteins, are key regulators of spatial compartmentalization in fungi and other eukaryotes. Septins form higher-order structures on fungal plasma membranes and are thought to contribute to the generation of cell asymmetries by acting as molecular scaffolds and forming diffusional barriers. Here we discuss the links between septins and polarized growth and consider molecular models for how septins contribute to cellular asymmetry in fungi.

Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells.

Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells.

Biological Significance of Photoreceptor Photocycle Length: VIVID Photocycle Governs the Dynamic VIVID-White Collar Complex Pool Mediating Photo-adaptation and Response to Changes in Light Intensity.

Most organisms on earth sense light through the use of chromophore-bearing photoreceptive proteins with distinct and characteristic photocycle lengths, yet the biological significance of this adduct decay length is neither understood nor has been tested. In the filamentous fungus Neurospora crassa VIVID (VVD) is a critical player in the process of photoadaptation, the attenuation of light-induced responses and the ability to maintain photosensitivity in response to changing light intensities. Detailed in vitro analysis of the photochemistry of the blue light sensing, FAD binding, LOV domain of VVD has revealed residues around the site of photo-adduct formation that influence the stability of the adduct state (light state), that is, altering the photocycle length. We have examined the biological significance of VVD photocycle length to photoadaptation and report that a double substitution mutant (vvdI74VI85V), previously shown to have a very fast light to dark state reversion in vitro, shows significantly reduced interaction with the White Collar Complex (WCC) resulting in a substantial photoadaptation defect. This reduced interaction impacts photoreceptor transcription factor WHITE COLLAR-1 (WC-1) protein stability when N. crassa is exposed to light: The fast-reverting mutant VVD is unable to form a dynamic VVD-WCC pool of the size required for photoadaptation as assayed both by attenuation of gene expression and the ability to respond to increasing light intensity. Additionally, transcription of the clock gene frequency (frq) is sensitive to changing light intensity in a wild-type strain but not in the fast photo-reversion mutant indicating that the establishment of this dynamic VVD-WCC pool is essential in general photobiology and circadian biology. Thus, VVD photocycle length appears sculpted to establish a VVD-WCC reservoir of sufficient size to sustain photoadaptation while maintaining sensitivity to changing light intensity. The great diversity in photocycle kinetics among photoreceptors may be viewed as reflecting adaptive responses to specific and salient tasks required by organisms to respond to different photic environments.

Quantitative spatial analysis of transcripts in multinucleate cells using single-molecule FISH.

mRNA positioning in the cell is important for diverse cellular functions and proper development of multicellular organisms. Single-molecule RNA FISH (smFISH) enables quantitative investigation of mRNA localization and abundance at the level of individual molecules in the context of cellular features. Details about spatial mRNA patterning at various times, in different genetic backgrounds, at different developmental stages, and under varied environmental conditions provide invaluable insights into the mechanisms and functions of spatial regulation. Here, we describe detailed methods for performing smFISH along with immunofluorescence for two large, multinucleate cell types: the fungus Ashbya gossypii and cultured mouse myotubes. We also put forward a semi-automated image processing tool that systematically detects mRNAs from smFISH data and statistically analyzes the spatial pattern of mRNAs using a customized MATLAB code. These protocols and image analysis tools can be adapted to a wide variety of transcripts and cell types for systematically and quantitatively analyzing mRNA distribution in three-dimensional space.

Septin assemblies form by diffusion-driven annealing on membranes.

Septins assemble into filaments and higher-order structures that act as scaffolds for diverse cell functions including cytokinesis, cell polarity, and membrane remodeling. Despite their conserved role in cell organization, little is known about how septin filaments elongate and are knitted together into higher-order assemblies. Using fluorescence correlation spectroscopy, we determined that cytosolic septins are in small complexes, suggesting that septin filaments are not formed in the cytosol. When the plasma membrane of live cells is monitored by total internal reflection fluorescence microscopy, we see that septin complexes of variable size diffuse in two dimensions. Diffusing septin complexes collide and make end-on associations to form elongated filaments and higher-order structures, an assembly process we call annealing. Septin assembly by annealing can be reconstituted in vitro on supported lipid bilayers with purified septin complexes. Using the reconstitution assay, we show that septin filaments are highly flexible, grow only from free filament ends, and do not exchange subunits in the middle of filaments. This work shows that annealing is a previously unidentified intrinsic property of septins in the presence of membranes and demonstrates that cells exploit this mechanism to build large septin assemblies.