Biology subjects

Riback, J. A.

Publications and source records attributed to Riback, J. A..

9 recordsLinked to original sources

bioRxiv2026

Adaptive energetic tuning of nucleolar phase separation regulates rRNA transport

Eukaryotic cells adjust biochemical pathways in response to environmental stressors, stalling energetically costly processes such as protein synthesis and ribosome biogenesis. Ribosome biogenesis occurs within the multiphase nucleolus that contains three layers roughly corresponding to ribosomal RNA (rRNA) transcription, processing, and assembly. While nucleolar perturbations induced by drug treatments and optogenetic nucleolar gelation alter nucleolar stability and hinder ribosome biogenesis, it remains unclear whether nucleolar properties are actively tuned in response to metabolic and growth cues. Here, we show that nucleolar phase separation is adjusted to physiological needs. Live-cell imaging of endogenously tagged NPM1 reveals a tight relationship between NPM1 partitioning and nutrient availability. In nutrient-deprived conditions, NPM1 levels in the nucleolus increase, indicative of stabilized phase separation and a more gel-like state. Indeed, we show that rRNA diffusion decreases and the nucleolar meshwork contracts. Furthermore, this change corresponds to decreased rRNA processing, suggesting that reduced transport properties prevent the release of immature ribosome subunits from the nucleolus. Mechanistically, we show that these changes are driven by ATP levels, which broadly affect the energetically expensive process of ribosome biogenesis. Upon restoration of ATP levels by nutrient reintroduction, nucleolar composition returns to normal within minutes, suggesting active regulation. Taken together, our findings reveal that the biophysical properties of the nucleolus are not fixed, but are actively remodeled by cellular energy levels, linking phase separation dynamics to metabolic control of ribosome biogenesis.

biophysics↗

bioRxiv2026

Mapping RNA structure assembly and remodeling in biomolecular condensates

Biomolecular condensates concentrate RNA and protein machinery that facilitate RNA processing, ribonucleoprotein assembly, and gene regulation. Some evidence supports that condensates can modulate RNA folding, but measuring RNA structure within native condensates remains an unsolved challenge. Here we introduce RAID-MaP, a strategy that combines APEX proximity labeling with dimethyl sulfate (DMS) chemical probing to measure RNA structure within defined subcellular compartments. We applied RAID-MaP to resolve late stages of ribosomal RNA (rRNA) folding within the granular component (GC) of the nucleolus, revealing that both the 18S and 28S rRNAs feature widespread differences in structure compared to assembled ribosomes consistent with ongoing folding of both secondary and tertiary structure. We further combined RAID-MaP with transcription inhibition to resolve kinetics of rRNA maturation. The maturation kinetics of both subunits progressed on comparable timescales but with characteristic domain-level ordering, with late GC-resident intermediates often becoming more protected than mature ribosomes suggestive of stabilization by nucleolar accessory factors. Perturbing this 28S assembly pathway using antisense oligonucleotides produces distinct nucleolar phenotypes depending on whether early versus late folding domains are disrupted, demonstrating a direct link between rRNA folding and phase separation. We additionally applied RAID-MaP to discover that the 7SK small nuclear RNA undergoes spatially regulated structural switching consistent with localized release of the transcription factor P-TEFb at sites of active transcription. Together, our results establish subcellular spatial control of RNA structure as a new dimension of RNA regulation.

biochemistry↗

bioRxiv2025

Nuclear Phase Separation Drives NPM1-mutant Acute Myeloid Leukemia

During cancer development, mutations promote gene expression changes that cause transformation. Leukemia is frequently associated with aberrant HOXA expression driven by translocations in nucleoporin genes or KMT2A, and mutations in NPM1. How disparate mutations converge on this regulatory pathway is not understood. Here we demonstrate that mutant NPM1 (NPM1c) forms nuclear condensates in multiple human cell lines, mouse models, and primary patient samples. We show NPM1c phase separation is necessary and sufficient to coordinate the recruitment of NUP98 and KMT2A to condensates. Through extensive mutagenesis and pharmacological destabilization of phase separation, we find that NPM1c condensates are necessary for regulating gene expression, promoting in vivo expansion, and maintaining the undifferentiated leukemic state. Finally, we show that nucleoporin and KMT2A fusion proteins form condensates that are biophysically indistinguishable from NPM1c condensates. Together, these data define a new condensate underlying leukemias that we term coordinating bodies (C-bodies), and propose C-bodies as a therapeutic vulnerability.

biophysics↗

bioRxiv2025

Nanometer condensate organization in live cells derived from partitioning measurements

Biomolecules self-organize into membrane-less organelles known as condensates that compartmentalize essential biochemical processes, such as ribosome biogenesis in the nucleolus1-3. Molecular dynamics within condensates are governed by chemical preferences and interaction networks that can imbue nanoscale structure4-7. Such organization is typically inferred from ensemble-averaged measurements, such as scattering and electron microscopy, which reveal molecular arrangements8-14. However, the complexity of cells obscures the interpretability of these techniques, limiting insight into condensate internal structure and roles in macromolecular assembly and transport. Here, we develop an approach to quantify the average microenvironment surrounding specific proteins within condensates in live cells, using thermodynamic principles to interpret the partitioning of designed protein probes. Using this approach, we find that condensates in cells, including the nucleolus, stress granule, and nuclear pore, exhibit spatial inhomogeneity, aligning with emerging views of condensates as networked fluids5,6,15-18. Within the nucleolus, we link spatial inhomogeneity to ribosome biogenesis, which progressively loosens the average local meshwork, facilitating transport of assembled ribosomal subunits. Within the nuclear pore, we find that transporters experience a weaker local meshwork than nucleoporins, consistent with the selective phase model19,20. Together, our approach uncovers a distinct mode of biomolecular control arising from nanoscale structure, which we term microenvironment coupling, whereby internal interaction landscapes shape transport to enable regulation and proofreading.

biophysics↗

bioRxiv2022

Sequential Activation and Local Unfolding Control Poly(A)-Binding Protein Condensation

Eukaryotic cells form biomolecular condensates to sense and adapt to their environment1,2. Poly(A)-binding protein (Pab1), a canonical stress granule marker3,4, condenses upon heat shock or starvation, promoting adaptation5. The molecular basis of condensation has remained elusive due to a dearth of techniques to probe structure directly in condensates. Here we apply hydrogen-deuterium exchange/mass spectrometry (HDX-MS) to investigate the molecular mechanism of Pab1s condensation. We find that Pab1s four RNA recognition motifs (RRMs) undergo different levels of partial unfolding upon condensation, and the changes are similar for thermal and pH stresses. Although structural heterogeneity is observed, the ability of MS to describe individual subpopulations allows us to identify which regions become partially unfolded and contribute to the condensates interaction network. Our data yield a clear molecular picture of Pab1s stress-triggered condensation, which we term sequential activation, wherein each RRM becomes activated at a temperature where it partially unfolds and associates with other likewise activated RRMs to form the condensate. This model thus implies that sequential activation is dictated by the underlying free energy surface, an effect we refer to as thermodynamic specificity. Our study represents a methodological advance for elucidating the interactions that drive biomolecular condensation that we anticipate will be widely applicable. Furthermore, our findings demonstrate how condensation can use thermodynamic specificity to perform an acute response to multiple, stresses, a potentially general mechanism for stress-responsive proteins.

biophysics↗

bioRxiv2022

A rich get richer effect governs intracellular condensate size distributions

Phase separation of biomolecules into condensates has emerged as a ubiquitous mechanism for intracellular organization and impacts many intracellular processes, including reaction pathways through clustering of enzymes and their intermediates. Precise and rapid spatiotemporal control of reactions by condensates requires tuning of their sizes. However, the physical processes that govern the distribution of condensate sizes remain unclear. Here, we utilize a combination of synthetic and native condensates to probe the underlying physical mechanisms determining condensate size. We find that both native nuclear speckles and FUS condensates formed with the synthetic Corelet system obey an exponential size distribution, which can be recapitulated in Monte Carlo simulations of fast nucleation followed by coalescence. By contrast, pathological aggregation of cytoplasmic Huntingtin polyQ protein exhibits a power-law size distribution, with an exponent of -1.41 {+/-} 0.02. These distinct behaviors reflect the relative importance of nucleation and coalescence kinetics: introducing continuous condensate nucleation into the Monte Carlo coarsening simulations gives rise to polyQ-like power-law behavior. We demonstrate that the emergence of power-law distributions under continuous nucleation reflects a "rich get richer" effect, whose extent may play a general role in the determination of condensate size distributions.

biophysics↗

bioRxiv2022

Viscoelastic RNA entanglement and advective flow underlie nucleolar form and function

The nucleolus facilitates transcription, processing, and assembly of ribosomal RNA (rRNA), the most abundant RNA in cells. Nucleolar function is facilitated by its multiphase liquid properties, but nucleolar fluidity and its connection to ribosome biogenesis remain unclear. Here, we used quantitative imaging, mathematical modeling, and pulse-chase nucleotide labelling to map nucleolar rRNA dynamics. Inconsistent with a purely diffusive process, rRNA steadily expands away from the transcriptional sites, moving in a slow (~1[A]/s), radially-directed fashion. This motion reflects the viscoelastic properties of a highly concentrated gel of entangled rRNA, whose constant polymerization drives steady outward flow. We propose a new viscoelastic rRNA release model, where nucleolar rRNA cleavage and processing reduce entanglement, fluidizing the nucleolar periphery to facilitate release of mature pre-ribosomal particles.

biophysics↗

bioRxiv2019

Composition dependent phase separation underlies directional flux through the nucleolus

Intracellular bodies such as nucleoli, Cajal bodies, and various signaling assemblies, represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions, particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions (IDRs), are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3-6 have led to the concept that a single fixed saturation concentration (Csat) is a defining feature of endogenous LLPS7-9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed Csat remains largely untested within living cells, where the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed Csat. As the concentration of individual components is varied, their partition coefficients change, in a manner that can be used to extract thermodynamic interaction energies, that we interpret within a framework we term polyphasic interaction thermodynamic analysis (PITA). We find that heterotypic interactions between protein and RNA components stabilize a variety of archetypal intracellular condensates, including the nucleolus, Cajal bodies, stress granules, and P bodies. These findings imply that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA processing condensates such as the nucleolus, this stoichiometric self-tuning manifests in selective exclusion of fully-assembled RNP complexes, providing a thermodynamic basis for vectorial ribosomal RNA (rRNA) flux out of the nucleolus. The PITA methodology is conceptually straightforward and readily implemented, and it can be broadly utilized to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deep understanding of the thermodynamics of multi-component intracellular phase behavior and its interplay with nonequilibrium activity characteristic of endogenous condensates.

biophysics↗

bioRxiv2018

Commonly-used FRET fluorophores promote collapse of an otherwise disordered protein.

The dimensions that unfolded proteins, including intrinsically disordered proteins (IDPs), adopt at low or no denaturant remains controversial. We recently developed an innovative analysis procedure for small-angle X-ray scattering (SAXS) profiles and found that even relatively hydrophobic IDPs remain nearly as expanded as the chemically denatured ensemble, rendering them significantly more expanded than generally inferred using fluorescence resonance energy transfer (FRET) measurements. We show here that fluorophores typical of those employed in FRET can contribute to this discrepancy. Specifically, we find that addition of Alexa488 to a normally expanded IDP causes contraction of its ensemble. In parallel, we also tested the recent suggestion that FRET and SAXS results can be reconciled if, in contrast to homopolymers, the radius of gyration (Rg) of an unfolded protein chain can vary independently from its end-to-end distance (Ree). To do so, we developed an analysis procedure that can accurately extract both Rg and Ree from SAXS profiles even if they are decoupled. Using this procedure, we find that Rg and Ree remain tightly coupled even for heteropolymeric IDPs. We thus conclude that, when combined with improved analysis procedures for both SAXS and FRET, fluorophore-driven interactions are sufficient to explain the preponderance of existing data regarding the nature of polypeptide chains unfolded in the absence of denaturant.

biophysics↗