The roles of FUS-RNA binding domain and low complexity domain in RNA-dependent phase separation.

Fused in sarcoma (FUS) is an archetypal phase separating protein asymmetrically divided into a low complexity domain (LCD) and an RNA binding domain (RBD). Here, we explore how the two domains contribute to RNA-dependent phase separation, RNA recognition, and multivalent complex formation. We find that RBD drives RNA-dependent phase separation but forms large and irregularly shaped droplets that are rescued by LCD in trans. Electrophoretic mobility shift assay (EMSA) and single-molecule fluorescence assays reveal that, while both LCD and RBD bind RNA, RBD drives RNA engagement and multivalent complex formation. While RBD alone exhibits delayed RNA recognition and a less dynamic RNP complex compared to full-length FUS, LCD in trans rescues full-length FUS activity. Likewise, cell-based data show RBD forms nucleolar condensates while LCD in trans rescues the diffuse nucleoplasm localization of full-length FUS. Our results point to a regulatory role of LCD in tuning the RNP interaction and buffering phase separation.

Linking folding dynamics and function of SAM/SAH riboswitches at the single molecule level.

Riboswitches are regulatory elements found in bacterial mRNAs that control downstream gene expression through ligand-induced conformational changes. Here, we used single-molecule FRET to map the conformational landscape of the translational SAM/SAH riboswitch and probe how co-transcriptional ligand-induced conformational changes affect its translation regulation function. Riboswitch folding is highly heterogeneous, suggesting a rugged conformational landscape that allows for sampling of the ligand-bound conformation even in the absence of ligand. The addition of ligand shifts the landscape, favoring the ligand-bound conformation. Mutation studies identified a key structural element, the pseudoknot helix, that is crucial for determining ligand-free conformations and their ligand responsiveness. We also investigated ribosomal binding site accessibility under two scenarios: pre-folding and co-transcriptional folding. The regulatory function of the SAM/SAH riboswitch involves kinetically favoring ligand binding, but co-transcriptional folding reduces this preference with a less compact initial conformation that exposes the Shine-Dalgarno sequence and takes min to redistribute to more compact conformations of the pre-folded riboswitch. Such slow equilibration decreases the effective ligand affinity. Overall, our study provides a deeper understanding of the complex folding process and how the riboswitch adapts its folding pattern in response to ligand, modulates ribosome accessibility and the role of co-transcriptional folding in these processes.

Nucleation and dissolution mechanism underlying amyotrophic lateral sclerosis/frontotemporal lobar dementia-linked fused in sarcoma condensates.

Fused in sarcoma (FUS) is a nuclear RNA-binding protein. Mutations in FUS lead to the mislocalization of FUS from the nucleus to the cytosol and formation of pathogenic aggregates in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD), yet with unknown molecular mechanisms. Using mutant and stress conditions, we visualized FUS localization and aggregate formation in cells. We used single-molecule pull-down (SiMPull) to quantify the native oligomerization states of wildtype (WT) and mutant FUS in cells. We demonstrate that the NLS mutants exhibited the highest oligomerization (>3) followed by other FUS mutants (>2) and WT FUS which is primarily monomeric. Strikingly, the mutant FUS oligomers are extremely stable and resistant to treatment by high salt, hexanediol, RNase, and Karyopherin-ß2 and only soluble in GdnHCl and SDS. We propose that the increased oligomerization units of mutant FUS and their high stability may contribute to ALS/FTLD pathogenesis.

RNA conformational propensities determine cellular activity.

Cellular processes are the product of interactions between biomolecules, which associate to form biologically active complexes1. These interactions are mediated by intermolecular contacts, which if disrupted, lead to alterations in cell physiology. Nevertheless, the formation of intermolecular contacts nearly universally requires changes in the conformations of the interacting biomolecules. As a result, binding affinity and cellular activity crucially depend both on the strength of the contacts and on the inherent propensities to form binding-competent conformational states2,3. Thus, conformational penalties are ubiquitous in biology and must be known in order to quantitatively model binding energetics for protein and nucleic acid interactions4,5. However, conceptual and technological limitations have hindered our ability to dissect and quantitatively measure how conformational propensities affect cellular activity. Here we systematically altered and determined the propensities for forming the protein-bound conformation of HIV-1 TAR RNA. These propensities quantitatively predicted the binding affinities of TAR to the RNA-binding region of the Tat protein and predicted the extent of HIV-1 Tat-dependent transactivation in cells. Our results establish the role of ensemble-based conformational propensities in cellular activity and reveal an example of a cellular process driven by an exceptionally rare and short-lived RNA conformational state.

Switch-like compaction of poly(ADP-ribose) upon cation binding.

Poly(ADP-ribose) (PAR) is a homopolymer of adenosine diphosphate ribose that is added to proteins as a posttranslational modification to regulate numerous cellular processes. PAR also serves as a scaffold for protein binding in macromolecular complexes, including biomolecular condensates. It remains unclear how PAR achieves specific molecular recognition. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) to evaluate PAR flexibility under different cation conditions. We demonstrate that, compared to RNA and DNA, PAR has a longer persistence length and undergoes a sharper transition from extended to compact states in physiologically relevant concentrations of various cations (Na+, Mg2+, Ca2+, and spermine4+). We show that the degree of PAR compaction depends on the concentration and valency of cations. Furthermore, the intrinsically disordered protein FUS also served as a macromolecular cation to compact PAR. Taken together, our study reveals the inherent stiffness of PAR molecules, which undergo switch-like compaction in response to cation binding. This study indicates that a cationic environment may drive recognition specificity of PAR.

Switch-like Compaction of Poly(ADP-ribose) Upon Cation Binding.

Poly(ADP-ribose) (PAR) is a homopolymer of adenosine diphosphate ribose that is added to proteins as a post-translational modification to regulate numerous cellular processes. PAR also serves as a scaffold for protein binding in macromolecular complexes, including biomolecular condensates. It remains unclear how PAR achieves specific molecular recognition. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) to evaluate PAR flexibility under different cation conditions. We demonstrate that, compared to RNA and DNA, PAR has a longer persistence length and undergoes a sharper transition from extended to compact states in physiologically relevant concentrations of various cations (Na + , Mg 2+ , Ca 2+ , and spermine). We show that the degree of PAR compaction depends on the concentration and valency of cations. Furthermore, the intrinsically disordered protein FUS also served as a macromolecular cation to compact PAR. Taken together, our study reveals the inherent stiffness of PAR molecules, which undergo switch-like compaction in response to cation binding. This study indicates that a cationic environment may drive recognition specificity of PAR. Significance: Poly(ADP-ribose) (PAR) is an RNA-like homopolymer that regulates DNA repair, RNA metabolism, and biomolecular condensate formation. Dysregulation of PAR results in cancer and neurodegeneration. Although discovered in 1963, fundamental properties of this therapeutically important polymer remain largely unknown. Biophysical and structural analyses of PAR have been exceptionally challenging due to the dynamic and repetitive nature. Here, we present the first single-molecule biophysical characterization of PAR. We show that PAR is stiffer than DNA and RNA per unit length. Unlike DNA and RNA which undergoes gradual compaction, PAR exhibits an abrupt switch-like bending as a function of salt concentration and by protein binding. Our findings points to unique physical properties of PAR that may drive recognition specificity for its function.

Single-Molecule Fluorescence Methods to Study Protein-RNA Interactions Underlying Biomolecular Condensates.

Many biomolecular condensates, including nucleoli and stress granules, form via dynamic multivalent protein-protein and protein-RNA interactions. These molecular interactions nucleate liquid-liquid phase separation (LLPS) and determine condensate properties, such as size and fluidity. Here, we outline the experimental procedures for single-molecule fluorescence experiments to probe protein-RNA interactions underlying LLPS. The experiments include single-molecule Förster (Fluorescence) resonance energy transfer (smFRET) to monitor protein-induced conformational changes in the RNA, protein-induced fluorescence enhancement (PIFE) to measure protein-RNA encounters, and single-molecule nucleation experiments to quantify the association and buildup of proteins on a nucleating RNA. Together, these experiments provide complementary approaches to elucidate a molecular view of the protein-RNA interactions that drive ribonucleoprotein condensate formation.

Poly(ADP-ribose) drives condensation of FUS via a transient interaction.

Poly(ADP-ribose) (PAR) is an RNA-like polymer that regulates an increasing number of biological processes. Dysregulation of PAR is implicated in neurodegenerative diseases characterized by abnormal protein aggregation, including amyotrophic lateral sclerosis (ALS). PAR forms condensates with FUS, an RNA-binding protein linked with ALS, through an unknown mechanism. Here, we demonstrate that a strikingly low concentration of PAR (1 nM) is sufficient to trigger condensation of FUS near its physiological concentration (1 µM), which is three orders of magnitude lower than the concentration at which RNA induces condensation (1 µM). Unlike RNA, which associates with FUS stably, PAR interacts with FUS transiently, triggering FUS to oligomerize into condensates. Moreover, inhibition of a major PAR-synthesizing enzyme, PARP5a, diminishes FUS condensation in cells. Despite their structural similarity, PAR and RNA co-condense with FUS, driven by disparate modes of interaction with FUS. Thus, we uncover a mechanism by which PAR potently seeds FUS condensation.

Understanding the characteristics of nonspecific binding of drug-like compounds to canonical stem-loop RNAs and their implications for functional cellular assays.

Identifying small molecules that selectively bind an RNA target while discriminating against all other cellular RNAs is an important challenge in RNA-targeted drug discovery. Much effort has been directed toward identifying drug-like small molecules that minimize electrostatic and stacking interactions that lead to nonspecific binding of aminoglycosides and intercalators to many stem-loop RNAs. Many such compounds have been reported to bind RNAs and inhibit their cellular activities. However, target engagement and cellular selectivity assays are not routinely performed, and it is often unclear whether functional activity directly results from specific binding to the target RNA. Here, we examined the propensities of three drug-like compounds, previously shown to bind and inhibit the cellular activities of distinct stem-loop RNAs, to bind and inhibit the cellular activities of two unrelated HIV-1 stem-loop RNAs: the transactivation response element (TAR) and the rev response element stem IIB (RREIIB). All compounds bound TAR and RREIIB in vitro, and two inhibited TAR-dependent transactivation and RRE-dependent viral export in cell-based assays while also exhibiting off-target interactions consistent with nonspecific activity. A survey of X-ray and NMR structures of RNA-small molecule complexes revealed that aminoglycosides and drug-like molecules form hydrogen bonds with functional groups commonly accessible in canonical stem-loop RNA motifs, in contrast to ligands that specifically bind riboswitches. Our results demonstrate that drug-like molecules can nonspecifically bind stem-loop RNAs most likely through hydrogen bonding and electrostatic interactions and reinforce the importance of assaying for off-target interactions and RNA selectivity in vitro and in cells when assessing novel RNA-binders.

Publisher Correction: High-performance virtual screening by targeting a high-resolution RNA dynamic ensemble.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Probing RNA Conformational Equilibria within the Functional Cellular Context.

Low-abundance short-lived non-native conformations referred to as excited states (ESs) are increasingly observed in vitro and implicated in the folding and biological activities of regulatory RNAs. We developed an approach for assessing the relative abundance of RNA ESs within the functional cellular context. Nuclear magnetic resonance (NMR) spectroscopy was used to estimate the degree to which substitution mutations bias conformational equilibria toward the inactive ES in vitro. The cellular activity of the ES-stabilizing mutants was used as an indirect measure of the conformational equilibria within the functional cellular context. Compensatory mutations that restore the ground-state conformation were used to control for changes in sequence. Using this approach, we show that the ESs of two regulatory RNAs from HIV-1, the transactivation response element (TAR) and the Rev response element (RRE), likely form in cells with abundances comparable to those measured in vitro, and their targeted stabilization may provide an avenue for developing anti-HIV therapeutics.

Demonstration that Small Molecules can Bind and Stabilize Low-abundance Short-lived RNA Excited Conformational States.

Many promising RNA drug targets have functions that require the formation of RNA-protein complexes, but inhibiting RNA-protein interactions can prove difficult using small molecules. Regulatory RNAs have been shown to transiently form excited conformational states (ESs) that remodel local aspects of secondary structure. In some cases, the ES conformation has been shown to be inactive and to be poorly recognized by protein binding partners. In these cases, specifically targeting and stabilizing the RNA ES using a small molecule provides a rational structure-based strategy for inhibiting RNA activity. However, this requires that a small molecule discriminates between two conformations of the same RNA to preferentially bind and stabilize the short-lived low-abundance ES relative to the long-lived more abundant ground state (GS). Here, we tested the feasibility of this approach by designing a mutant that inverts the conformational equilibrium of the HIV-1 transactivation response element (TAR) RNA, such that the native GS conformation becomes a low-abundance ES. Using this mutant and NMR chemical shift mapping experiments, we show that argininamide, a ligand mimic of TAR's cognate protein binding partner Tat, is able to restore a native-like conformation by preferentially binding and stabilizing the transient and low-populated ES. A synthetic small molecule optimized to bind the TAR GS also partially stabilized the ES, whereas an aminoglycoside molecule that binds RNAs nonspecifically did not preferentially stabilize the ES to a similar extent. These results support the feasibility of inhibiting RNA activity using small molecules that preferentially bind and stabilize the ES.

The roles of structural dynamics in the cellular functions of RNAs.

RNAs fold into 3D structures that range from simple helical elements to complex tertiary structures and quaternary ribonucleoprotein assemblies. The functions of many regulatory RNAs depend on how their 3D structure changes in response to a diverse array of cellular conditions. In this Review, we examine how the structural characterization of RNA as dynamic ensembles of conformations, which form with different probabilities and at different timescales, is improving our understanding of RNA function in cells. We discuss the mechanisms of gene regulation by microRNAs, riboswitches, ribozymes, post-transcriptional RNA modifications and RNA-binding proteins, and how the cellular environment and processes such as liquid-liquid phase separation may affect RNA folding and activity. The emerging RNA-ensemble-function paradigm is changing our perspective and understanding of RNA regulation, from in vitro to in vivo and from descriptive to predictive.

High-performance virtual screening by targeting a high-resolution RNA dynamic ensemble.

Dynamic ensembles hold great promise in advancing RNA-targeted drug discovery. Here we subjected the transactivation response element (TAR) RNA from human immunodeficiency virus type-1 to experimental high-throughput screening against ~100,000 drug-like small molecules. Results were augmented with 170 known TAR-binding molecules and used to generate sublibraries optimized for evaluating enrichment when virtually screening a dynamic ensemble of TAR determined by combining NMR spectroscopy data and molecular dynamics simulations. Ensemble-based virtual screening scores molecules with an area under the receiver operator characteristic curve of ~0.85-0.94 and with ~40-75% of all hits falling within the top 2% of scored molecules. The enrichment decreased significantly for ensembles generated from the same molecular dynamics simulations without input NMR data and for other control ensembles. The results demonstrate that experimentally determined RNA ensembles can significantly enrich libraries with true hits and that the degree of enrichment is dependent on the accuracy of the ensemble.

Amiloride as a new RNA-binding scaffold with activity against HIV-1 TAR.

Diversification of RNA-targeted scaffolds offers great promise in the search for selective ligands of therapeutically relevant RNA such as HIV-1 TAR. We herein report the establishment of amiloride as a novel RNA-binding scaffold along with synthetic routes for combinatorial C(5)- and C(6)-diversification. Iterative modifications at the C(5)- and C(6)- positions yielded derivative 24, which demonstrated a 100-fold increase in activity over the parent dimethylamiloride in peptide displacement assays. NMR chemical shift mapping was performed using the 2D SOFAST- [1H-13C] HMQC NMR method, which allowed for facile and rapid evaluation of binding modes for all library members. Cheminformatic analysis revealed distinct differences between selective and non-selective ligands. In this study, we evolved dimethylamiloride from a weak TAR ligand to one of the tightest binding selective TAR ligands reported to date through a novel combination of synthetic methods and analytical techniques. We expect these methods to allow for rapid library expansion and tuning of the amiloride scaffold for a range of RNA targets and for SOFAST NMR to allow unprecedented evaluation of small molecule:RNA interactions.

Resolving sugar puckers in RNA excited states exposes slow modes of repuckering dynamics.

Recent studies have shown that RNAs exist in dynamic equilibrium with short-lived low-abundance 'excited states' that form by reshuffling base pairs in and around non-canonical motifs. These conformational states are proposed to be rich in non-canonical motifs and to play roles in the folding and regulatory functions of non-coding RNAs but their structure proves difficult to characterize given their transient nature. Here, we describe an approach for determining sugar pucker conformation in RNA excited states through nuclear magnetic resonance measurements of C1΄ and C4΄ rotating frame spin relaxation (R1ρ) in uniformly 13C/15N labeled RNA samples. Application to HIV-1 TAR exposed slow modes of sugar repuckering dynamics at the µs and ms timescale accompanying transitions between non-helical (C2΄-endo) to helical (C3΄-endo) conformations during formation of two distinct excited states. In contrast, we did not obtain any evidence for slow sugar repuckering dynamics for nucleotides in a variety of structural contexts that do not undergo non-helical to helical transitions. Our results outline a route for significantly improving the conformational characterization of RNA excited states and suggest that slow modes of repuckering dynamics gated by transient changes in secondary structure are quite common in RNA.

An RNA tertiary switch by modifying how helices are tethered.

A viral tRNA-like structure has evolved a unique strategy to undergo a tertiary structure conformational switch that may help regulate viral regulation.