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.

CAG-Repeat RNA Hairpin Folding and Recruitment to Nuclear Speckles with a Pivotal Role of ATP as a Cosolute.

A hallmark of Huntington's disease (HD) is a prolonged polyglutamine sequence in the huntingtin protein and, correspondingly, an expanded cytosine, adenine, and guanine (CAG) triplet repeat region in the mRNA. A majority of studies investigating disease pathology were concerned with toxic huntingtin protein, but the mRNA moved into focus due to its recruitment to RNA foci and emerging novel therapeutic approaches targeting the mRNA. A hallmark of CAG-RNA is that it forms a stable hairpin in vitro which seems to be crucial for specific protein interactions. Using in-cell folding experiments, we show that the CAG-RNA is largely destabilized in cells compared to dilute buffer solutions but remains folded in the cytoplasm and nucleus. Surprisingly, we found the same folding stability in the nucleoplasm and in nuclear speckles under physiological conditions suggesting that CAG-RNA does not undergo a conformational transition upon recruitment to the nuclear speckles. We found that the metabolite adenosine triphosphate (ATP) plays a crucial role in promoting unfolding, enabling its recruitment to nuclear speckles and preserving its mobility. Using in vitro experiments and molecular dynamics simulations, we found that the ATP effects can be attributed to a direct interaction of ATP with the nucleobases of the CAG-RNA rather than ATP acting as "a fuel" for helicase activity. ATP-driven changes in CAG-RNA homeostasis could be disease-relevant since mitochondrial function is affected in HD disease progression leading to a decline in cellular ATP levels.

An amphipathic helix in Brl1 is required for nuclear pore complex biogenesis in S. cerevisiae.

The nuclear pore complex (NPC) is the central portal for macromolecular exchange between the nucleus and cytoplasm. In all eukaryotes, NPCs assemble into an intact nuclear envelope (NE) during interphase, but the process of NPC biogenesis remains poorly characterized. Furthermore, little is known about how NPC assembly leads to the fusion of the outer and inner NE, and no factors have been identified that could trigger this event. Here, we characterize the transmembrane protein Brl1 as an NPC assembly factor required for NE fusion in budding yeast. Brl1 preferentially associates with NPC assembly intermediates and its depletion halts NPC biogenesis, leading to NE herniations that contain inner and outer ring nucleoporins but lack the cytoplasmic export platform. Furthermore, we identify an essential amphipathic helix in the luminal domain of Brl1 that mediates interactions with lipid bilayers. Mutations in this amphipathic helix lead to NPC assembly defects, and cryo-electron tomography analyses reveal multilayered herniations of the inner nuclear membrane with NPC-like structures at the neck, indicating a failure in NE fusion. Taken together, our results identify a role for Brl1 in NPC assembly and suggest a function of its amphipathic helix in mediating the fusion of the inner and outer nuclear membranes.

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.

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.

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.

The cellular environment shapes the nuclear pore complex architecture.

Nuclear pore complexes (NPCs) create large conduits for cargo transport between the nucleus and cytoplasm across the nuclear envelope (NE)1-3. These multi-megadalton structures are composed of about thirty different nucleoporins that are distributed in three main substructures (the inner, cytoplasmic and nucleoplasmic rings) around the central transport channel4-6. Here we use cryo-electron tomography on DLD-1 cells that were prepared using cryo-focused-ion-beam milling to generate a structural model for the human NPC in its native environment. We show that-compared with previous human NPC models obtained from purified NEs-the inner ring in our model is substantially wider; the volume of the central channel is increased by 75% and the nucleoplasmic and cytoplasmic rings are reorganized. Moreover, the NPC membrane exhibits asymmetry around the inner-ring complex. Using targeted degradation of Nup96, a scaffold nucleoporin of the cytoplasmic and nucleoplasmic rings, we observe the interdependence of each ring in modulating the central channel and maintaining membrane asymmetry. Our findings highlight the inherent flexibility of the NPC and suggest that the cellular environment has a considerable influence on NPC dimensions and architecture.

Dead or alive: DEAD-box ATPases as regulators of ribonucleoprotein complex condensation.

DEAD-box ATPase proteins are found in all clades of life and have been associated with a diverse array of RNA-processing reactions in eukaryotes, bacteria and archaea. Their highly conserved core enables them to bind RNA, often in an ATP-dependent manner. In the course of the ATP hydrolysis cycle, they undergo conformational rearrangements, which enable them to unwind short RNA duplexes or remodel RNA-protein complexes. Thus, they can function as RNA helicases or chaperones. However, when their conformation is locked, they can also clamp RNA and create ATP-dependent platforms for the formation of higher-order ribonucleoprotein complexes. Recently, it was shown that DEAD-box ATPases globally regulate the phase-separation behavior of RNA-protein complexes in vitro and control the dynamics of RNA-containing membraneless organelles in both pro- and eukaryotic cells. A role of these enzymes as regulators of RNA-protein condensates, or 'condensases', suggests a unifying view of how the biochemical activities of DEAD-box ATPases are used to keep cellular condensates dynamic and 'alive', and how they regulate the composition and fate of ribonucleoprotein complexes in different RNA processing steps.

Maturation Kinetics of a Multiprotein Complex Revealed by Metabolic Labeling.

All proteins interact with other cellular components to fulfill their function. While tremendous progress has been made in the identification of protein complexes, their assembly and dynamics remain difficult to characterize. Here, we present a high-throughput strategy to analyze the native assembly kinetics of protein complexes. We apply our approach to characterize the co-assembly for 320 pairs of nucleoporins (NUPs) constituting the ≈50 MDa nuclear pore complex (NPC) in yeast. Some NUPs co-assemble fast via rapid exchange whereas others require lengthy maturation steps. This reveals a hierarchical principle of NPC biogenesis where individual subcomplexes form on a minute timescale and then co-assemble from center to periphery in a ∼1 h-long maturation process. Intriguingly, the NUP Mlp1 stands out as joining very late and associating preferentially with aged NPCs. Our approach is readily applicable beyond the NPC, making it possible to analyze the intracellular dynamics of a variety of multiprotein assemblies.

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.

ß-Oxidation and autophagy are critical energy providers during acute glucose depletion in Saccharomyces cerevisiae.

The ability to tolerate and thrive in diverse environments is paramount to all living organisms, and many organisms spend a large part of their lifetime in starvation. Upon acute glucose starvation, yeast cells undergo drastic physiological and metabolic changes and reestablish a constant-although lower-level of energy production within minutes. The molecules that are rapidly metabolized to fuel energy production under these conditions are unknown. Here, we combine metabolomics and genetics to characterize the cells' response to acute glucose depletion and identify pathways that ensure survival during starvation. We show that the ability to respire is essential for maintaining the energy status and to ensure viability during starvation. Measuring the cells' immediate metabolic response, we find that central metabolites drastically deplete and that the intracellular AMP-to-ATP ratio strongly increases within 20 to 30 s. Furthermore, we detect changes in both amino acid and lipid metabolite levels. Consistent with this, both bulk autophagy, a process that frees amino acids, and lipid degradation via ß-oxidation contribute in parallel to energy maintenance upon acute starvation. In addition, both these pathways ensure long-term survival during starvation. Thus, our results identify bulk autophagy and ß-oxidation as important energy providers during acute glucose starvation.

The RNA export factor Mex67 functions as a mobile nucleoporin.

The RNA export factor Mex67 is essential for the transport of mRNA through the nuclear pore complex (NPC) in yeast, but the molecular mechanism of this export process remains poorly understood. Here, we use quantitative fluorescence microscopy techniques in live budding yeast cells to investigate how Mex67 facilitates mRNA export. We show that Mex67 exhibits little interaction with mRNA in the nucleus and localizes to the NPC independently of mRNA, occupying a set of binding sites offered by FG repeats in the NPC. The ATPase Dbp5, which is thought to remove Mex67 from transcripts, does not affect the interaction of Mex67 with the NPC. Strikingly, we find that the essential function of Mex67 is spatially restricted to the NPC since a fusion of Mex67 to the nucleoporin Nup116 rescues a deletion of MEX67 Thus, Mex67 functions as a mobile NPC component, which receives mRNA export substrates in the central channel of the NPC to facilitate their translocation to the cytoplasm.

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.

Mapping the native organization of the yeast nuclear pore complex using nuclear radial intensity measurements.

Selective transport across the nuclear envelope (NE) is mediated by the nuclear pore complex (NPC), a massive ∼100-MDa assembly composed of multiple copies of ∼30 nuclear pore proteins (Nups). Recent advances have shed light on the composition and structure of NPCs, but approaches that could map their organization in live cells are still lacking. Here, we introduce an in vivo method to perform nuclear radial intensity measurements (NuRIM) using fluorescence microscopy to determine the average position of NE-localized proteins along the nucleocytoplasmic transport axis. We apply NuRIM to study the organization of the NPC and the mobile transport machinery in budding yeast. This reveals a unique snapshot of the intact yeast NPC and identifies distinct steady-state localizations for various NE-associated proteins and nuclear transport factors. We find that the NPC architecture is robust against compositional changes and could also confirm that in contrast to Chlamydomonas reinhardtii, the scaffold Y complex is arranged symmetrically in the yeast NPC. Furthermore, NuRIM was applied to probe the orientation of intrinsically disordered FG-repeat segments, providing insight into their roles in selective NPC permeability and structure.

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.

Small and Large Ribosomal Subunit Deficiencies Lead to Distinct Gene Expression Signatures that Reflect Cellular Growth Rate.

Levels of the ribosome, the conserved molecular machine that mediates translation, are tightly linked to cellular growth rate. In humans, ribosomopathies are diseases associated with cell-type-specific pathologies and reduced ribosomal protein (RP) levels. Because gene expression defects resulting from ribosome deficiency have not yet been experimentally defined, we systematically probed mRNA, translation, and protein signatures that were either unlinked from or linked to cellular growth rate in RP-deficient yeast cells. Ribosome deficiency was associated with altered translation of gene subclasses, and profound general secondary effects of RP loss on the spectrum of cellular mRNAs were seen. Among these effects, growth-defective 60S mutants increased synthesis of proteins involved in proteasome-mediated degradation, whereas 40S mutants accumulated mature 60S subunits and increased translation of ribosome biogenesis genes. These distinct signatures of protein synthesis suggest intriguing and currently mysterious differences in the cellular consequences of deficiency for small and large ribosomal subunits.

Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability.

The cytoplasmic abundance of mRNAs is strictly controlled through a balance of production and degradation. Whereas the control of mRNA synthesis through transcription has been well characterized, less is known about the regulation of mRNA turnover, and a consensus model explaining the wide variations in mRNA decay rates remains elusive. Here, we combine non-invasive transcriptome-wide mRNA production and stability measurements with selective and acute perturbations to demonstrate that mRNA degradation is tightly coupled to the regulation of translation, and that a competition between translation initiation and mRNA decay -but not codon optimality or elongation- is the major determinant of mRNA stability in yeast. Our refined measurements also reveal a remarkably dynamic transcriptome with an average mRNA half-life of only 4.8 min - much shorter than previously thought. Furthermore, global mRNA destabilization by inhibition of translation initiation induces a dose-dependent formation of processing bodies in which mRNAs can decay over time.