The Story of RNA Unfolded: The Molecular Function of DEAD- and DExH-Box ATPases and Their Complex Relationship with Membraneless Organelles.

DEAD- and DExH-box ATPases (DDX/DHXs) are abundant and highly conserved cellular enzymes ubiquitously involved in RNA processing. By remodeling RNA-RNA and RNA-protein interactions, they often function as gatekeepers that control the progression of diverse RNA maturation steps. Intriguingly, most DDX/DHXs localize to membraneless organelles (MLOs) such as nucleoli, nuclear speckles, stress granules, or processing bodies. Recent findings suggest not only that localization to MLOs can promote interaction between DDX/DHXs and their targets but also that DDX/DHXs are key regulators of MLO formation and turnover through their condensation and ATPase activity.In this review, we describe the molecular function of DDX/DHXs in ribosome biogenesis, messenger RNA splicing, export, translation, and storage or decay as well as their association with prominent MLOs. We discuss how the enzymatic function of DDX/DHXs in RNA processing is linked to DDX/DHX condensation, the accumulation of ribonucleoprotein particles and MLO dynamics. Future research will reveal how these processes orchestrate the RNA life cycle in MLO space and DDX/DHX time.

Glucose stress causes mRNA retention in nuclear Nab2 condensates.

Nuclear mRNA export via nuclear pore complexes is an essential step in eukaryotic gene expression. Although factors involved in mRNA transport have been characterized, a comprehensive mechanistic understanding of this process and its regulation is lacking. Here, we use single-RNA imaging in yeast to show that cells use mRNA retention to control mRNA export during stress. We demonstrate that, upon glucose withdrawal, the essential RNA-binding factor Nab2 forms RNA-dependent condensate-like structures in the nucleus. This coincides with a reduced abundance of the DEAD-box ATPase Dbp5 at the nuclear pore. Depleting Dbp5, and consequently blocking mRNA export, is necessary and sufficient to trigger Nab2 condensation. The state of Nab2 condensation influences the extent of nuclear mRNA accumulation and can be recapitulated in vitro, where Nab2 forms RNA-dependent liquid droplets. We hypothesize that cells use condensation to regulate mRNA export and control gene expression during stress.

DEAD-box ATPases as regulators of biomolecular condensates and membrane-less organelles.

RNA-dependent DEAD-box ATPases (DDXs) are emerging as major regulators of RNA-containing membrane-less organelles (MLOs). On the one hand, oligomerizing DDXs can promote condensate formation 'in cis', often using RNA as a scaffold. On the other hand, DDXs can disrupt RNA-RNA and RNA-protein interactions and thereby 'in trans' remodel the multivalent interactions underlying MLO formation. In this review, we discuss the best studied examples of DDXs modulating MLOs in cis and in trans. Further, we illustrate how this contributes to the dynamic assembly and turnover of MLOs which might help cells to modulate RNA sequestration and processing in a temporal and spatial manner.

Probing Liquid-Liquid Phase Separation of RNA-Binding Proteins In Vitro and In Vivo.

Biomolecular condensates and the concept of liquid-liquid phase separation (LLPS) have transformed cell biology in recent years. Condensates organize cellular content and compartmentalize biochemical reactions, in particular many processes involving RNA. This protocol is aimed at readers new to the LLPS field who want to test their protein or cellular structure of interest. We describe the basic principles of liquid-liquid phase separation, and outline initial approaches-both in vitro and in yeast cells-for the characterization of a candidate cellular condensate. First, we focus on strategies to purify phase-separating proteins and to reconstitute condensates from recombinant proteins in vitro for observation by light microscopy. Second, we describe in vivo experiments (including fluorescence recovery after photobleaching (FRAP) microscopy and 1,6-Hexanediol treatment) to test whether a subcellular structure displays liquid-like behavior in cells.

Dynamic arrest and aging of biomolecular condensates are modulated by low-complexity domains, RNA and biochemical activity.

Biomolecular condensates require suitable control of material properties for their function. Here we apply Differential Dynamic Microscopy (DDM) to probe the material properties of an in vitro model of processing bodies consisting of out-of-equilibrium condensates formed by the DEAD-box ATPase Dhh1 in the presence of ATP and RNA. By applying this single-droplet technique we show that condensates within the same population exhibit a distribution of material properties, which are regulated on several levels. Removal of the low-complexity domains (LCDs) of the protein decreases the fluidity of the condensates. Structured RNA leads to a larger fraction of dynamically arrested condensates with respect to unstructured polyuridylic acid (polyU). Promotion of the enzymatic ATPase activity of Dhh1 reduces aging of the condensates and the formation of arrested structures, indicating that biochemical activity and material turnover can maintain fluid-like properties over time.

Characterization of RNA content in individual phase-separated coacervate microdroplets.

Condensates formed by complex coacervation are hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of biological condensates and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and to which extend single condensates differ in their RNA composition. To address this, we developed an approach to read the RNA content from single synthetic and protein-based condensates using high-throughput sequencing. We find that certain RNAs efficiently accumulate in condensates. These RNAs are strongly enriched in sequence motifs which show high sequence similarity to short interspersed elements (SINEs). We observe similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution.

The Role of DEAD-Box ATPases in Gene Expression and the Regulation of RNA-Protein Condensates.

DEAD-box ATPases constitute a very large protein family present in all cells, often in great abundance. From bacteria to humans, they play critical roles in many aspects of RNA metabolism, and due to their widespread importance in RNA biology, they have been characterized in great detail at both the structural and biochemical levels. DEAD-box proteins function as RNA-dependent ATPases that can unwind short duplexes of RNA, remodel ribonucleoprotein (RNP) complexes, or act as clamps to promote RNP assembly. Yet, it often remains enigmatic how individual DEAD-box proteins mechanistically contribute to specific RNA-processing steps. Here, we review the role of DEAD-box ATPases in the regulation of gene expression and propose that one common function of these enzymes is in the regulation of liquid-liquid phase separation of RNP condensates.

RNA-bound PGC-1α controls gene expression in liquid-like nuclear condensates.

Plasticity of cells, tissues, and organs is controlled by the coordinated transcription of biological programs. However, the mechanisms orchestrating such context-specific transcriptional networks mediated by the dynamic interplay of transcription factors and coregulators are poorly understood. The peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a prototypical master regulator of adaptive transcription in various cell types. We now uncovered a central function of the C-terminal domain of PGC-1α to bind RNAs and assemble multiprotein complexes including proteins that control gene transcription and RNA processing. These interactions are important for PGC-1α recruitment to chromatin in transcriptionally active liquid-like nuclear condensates. Notably, such a compartmentalization of active transcription mediated by liquid-liquid phase separation was observed in mouse and human skeletal muscle, revealing a mechanism by which PGC-1α regulates complex transcriptional networks. These findings provide a broad conceptual framework for context-dependent transcriptional control of phenotypic adaptations in metabolically active tissues.

Membraneless organelles: phasing out of equilibrium.

Over the past years, liquid-liquid phase separation (LLPS) has emerged as a ubiquitous principle of cellular organization implicated in many biological processes ranging from gene expression to cell division. The formation of biological condensates, like the nucleolus or stress granules, by LLPS is at its core a thermodynamic equilibrium process. However, life does not operate at equilibrium, and cells have evolved multiple strategies to keep condensates in a non-equilibrium state. In this review, we discuss how these non-equilibrium drivers counteract solidification and potentially detrimental aggregation, and at the same time enable biological condensates to perform work and control the flux of substrates and information in a spatial and temporal manner.

DEAD-box ATPases are global regulators of phase-separated organelles.

The ability of proteins and nucleic acids to undergo liquid-liquid phase separation has recently emerged as an important molecular principle of how cells rapidly and reversibly compartmentalize their components into membrane-less organelles such as the nucleolus, processing bodies or stress granules1,2. How the assembly and turnover of these organelles are controlled, and how these biological condensates selectively recruit or release components are poorly understood. Here we show that members of the large and highly abundant family of RNA-dependent DEAD-box ATPases (DDXs)3 are regulators of RNA-containing phase-separated organelles in prokaryotes and eukaryotes. Using in vitro reconstitution and in vivo experiments, we demonstrate that DDXs promote phase separation in their ATP-bound form, whereas ATP hydrolysis induces compartment turnover and release of RNA. This mechanism of membrane-less organelle regulation reveals a principle of cellular organization that is conserved from bacteria to humans. Furthermore, we show that DDXs control RNA flux into and out of phase-separated organelles, and thus propose that a cellular network of dynamic, DDX-controlled compartments establishes biochemical reaction centres that provide cells with spatial and temporal control of various RNA-processing steps, which could regulate the composition and fate of ribonucleoprotein particles.

Dynamics of Synthetic Membraneless Organelles in Microfluidic Droplets.

Cells can form membraneless organelles by liquid-liquid phase separation. As these organelles are highly dynamic, it is crucial to understand the kinetics of these phase transitions. Here, we use droplet-based microfluidics to mix reagents by chaotic advection and observe nucleation, growth, and coarsening in volumes comparable to cells (pL) and on timescales of seconds. We apply this platform to analyze the dynamics of synthetic organelles formed by the DEAD-box ATPase Dhh1 and RNA, which are associated with the formation of processing bodies in yeast. We show that the timescale of phase separation decreases linearly as the volume of the compartment increases. Moreover, the synthetic organelles coarsen into one single droplet via gravity-induced coalescence, which can be arrested by introducing a hydrogel matrix that mimics the cytoskeleton. This approach is an attractive platform to investigate the dynamics of compartmentalization in artificial cells.

Pat1 promotes processing body assembly by enhancing the phase separation of the DEAD-box ATPase Dhh1 and RNA.

Processing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid-liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. Previously, we identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo (Mugler et al., 2016). Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.

Multifunctional Protein Materials and Microreactors using Low Complexity Domains as Molecular Adhesives.

Recent findings indicate that a class of disordered amino acid sequences promotes functional phase transition of biomolecules in nature. Such sequences consist of low complexity domains (LCDs) that are rich in specific amino acids. In this work, we exploit these sequences by conjugating them to soluble globular domains to develop molecular adhesives that enable sensitive, controlled self-assembly of these proteins into supramolecular architectures. In particular, we used the enzyme adenylate kinase and the green fluorescent protein as soluble domains, and we show that the addition of low complexity regions induces the formation of protein particles via a multistep process. This multistep pathway involves an initial liquid-liquid phase transition, which creates protein-rich droplets that mature into protein aggregates over time. These protein aggregates consist of permeable structures that maintain activity and release active soluble proteins. We show that the LCDs dictate specific noncovalent intermolecular interactions and phase properties that are largely independent of the given globular domain. We further demonstrate that this feature, together with the dynamic state of the initial dense liquid phase, allows one to directly assemble different globular domains within the same architecture, thereby enabling the generation of both static multifunctional biomaterials and dynamic microscale bioreactors.

ATPase activity of the DEAD-box protein Dhh1 controls processing body formation.

Translational repression and mRNA degradation are critical mechanisms of posttranscriptional gene regulation that help cells respond to internal and external cues. In response to certain stress conditions, many mRNA decay factors are enriched in processing bodies (PBs), cellular structures involved in degradation and/or storage of mRNAs. Yet, how cells regulate assembly and disassembly of PBs remains poorly understood. Here, we show that in budding yeast, mutations in the DEAD-box ATPase Dhh1 that prevent ATP hydrolysis, or that affect the interaction between Dhh1 and Not1, the central scaffold of the CCR4-NOT complex and an activator of the Dhh1 ATPase, prevent PB disassembly in vivo. Intriguingly, this process can be recapitulated in vitro, since recombinant Dhh1 and RNA, in the presence of ATP, phase-separate into liquid droplets that rapidly dissolve upon addition of Not1. Our results identify the ATPase activity of Dhh1 as a critical regulator of PB formation.

Structural basis of histone H2A-H2B recognition by the essential chaperone FACT.

Facilitates chromatin transcription (FACT) is a conserved histone chaperone that reorganizes nucleosomes and ensures chromatin integrity during DNA transcription, replication and repair. Key to the broad functions of FACT is its recognition of histones H2A-H2B (ref. 2). However, the structural basis for how histones H2A-H2B are recognized and how this integrates with the other functions of FACT, including the recognition of histones H3-H4 and other nuclear factors, is unknown. Here we reveal the crystal structure of the evolutionarily conserved FACT chaperone domain Spt16M from Chaetomium thermophilum, in complex with the H2A-H2B heterodimer. A novel 'U-turn' motif scaffolded onto a Rtt106-like module embraces the α1 helix of H2B. Biochemical and in vivo assays validate the structure and dissect the contribution of histone tails and H3-H4 towards Spt16M binding. Furthermore, we report the structure of the FACT heterodimerization domain that connects FACT to replicative polymerases. Our results show that Spt16M makes several interactions with histones, which we suggest allow the module to invade the nucleosome gradually and block the strongest interaction of H2B with DNA. FACT would thus enhance 'nucleosome breathing' by re-organizing the first 30 base pairs of nucleosomal histone-DNA contacts. Our snapshot of the engagement of the chaperone with H2A-H2B and the structures of all globular FACT domains enable the high-resolution analysis of the vital chaperoning functions of FACT, shedding light on how the complex promotes the activity of enzymes that require nucleosome reorganization.

Catch me if you can: how the histone chaperone FACT capitalizes on nucleosome breathing.

Nucleosomes confer a barrier to processes that require access to the eukaryotic genome such as transcription, DNA replication and repair. A variety of ATP-dependent nucleosome remodeling machines and ATP-independent histone chaperones facilitate nucleosome dynamics by depositing or evicting histones and unwrapping the DNA. It is clear that remodeling machines can use the energy from ATP to actively destabilize, translocate or disassemble nucleosomes. But how do ATP-independent histone chaperones, which "merely" bind histones, contribute to this process? Using our recent structural analysis of the conserved and essential eukaryotic histone chaperone FACT in complex with histones H2A-H2B as an example, we suggest that FACT capitalizes on transiently exposed surfaces of the nucleosome. By binding these surfaces, FACT stabilizes thermodynamically unfavorable intermediates of the intrinsically dynamic nucleosome particle. This makes the nucleosome permissive to DNA and RNA polymerases, providing temporary access, passage, and read-out.

The chaperone-histone partnership: for the greater good of histone traffic and chromatin plasticity.

Histones are highly positively charged proteins that wrap our genome. Their surface properties also make them prone to nonspecific interactions and aggregation. A class of proteins known as histone chaperones is dedicated to safeguard histones by aiding their proper incorporation into nucleosomes. Histone chaperones facilitate ordered nucleosome assembly and disassembly reactions through the formation of semi-stable histone-chaperone intermediates without requiring ATP, but merely providing a complementary protein surface for histones to dynamically interact with. Recurrent 'chaperoning' mechanisms involve the masking of the histone's positive charge and the direct blocking of crucial histone surface sites, including those required for H3-H4 tetramerization or the binding of nucleosomal DNA. This shielding prevents histones from engaging in premature or unwanted interactions with nucleic acids and other cellular components. In this review, we analyze recent structural studies on chaperone-histone interactions and discuss the implications of this vital partnership for nucleosome assembly and disassembly pathways.

A mitotic beacon reveals its nucleosome anchor.

Mitosis, nuclear envelope formation, and nucleocytoplasmic transport require chromosomes to identify themselves by enriching Ran-GTP around the chromatin fiber. In a recent Nature report, Makde et al. (2010) describe the structure of the Ran activator RCC1 anchored onto nucleosomes.

Alternative chromatin structures of the 35S rRNA genes in Saccharomyces cerevisiae provide a molecular basis for the selective recruitment of RNA polymerases I and II.

In all eukaryotes, a specialized enzyme, RNA polymerase I (Pol I), is dedicated to transcribe the 35S rRNA gene from a multicopy gene cluster, the ribosomal DNA (rDNA). In certain Saccharomyces cerevisiae mutants, 35S rRNA genes can be transcribed by RNA polymerase II (Pol II). In these mutants, rDNA silencing of Pol II transcription is impaired. It has been speculated that upstream activating factor (UAF), which binds to a specific DNA element within the Pol I promoter, plays a crucial role in forming chromatin structures responsible for polymerase specificity and silencing at the rDNA locus. We therefore performed an in-depth analysis of chromatin structure and composition in different mutant backgrounds. We demonstrate that chromatin architecture of the entire Pol I-transcribed region is substantially altered in the absence of UAF, allowing RNA polymerases II and III to access DNA elements flanking a Pol promoter-proximal Reb1 binding site. Furthermore, lack of UAF leads to the loss of Sir2 from rDNA, correlating with impaired Pol II silencing. This analysis of rDNA chromatin provides a molecular basis, explaining many phenotypes observed in previous genetic analyses.