Publications by authors named "Laura R Ganser"

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.

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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.

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Cellular processes are the product of interactions between biomolecules, which associate to form biologically active complexes. 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.

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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.

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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.

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Unlabelled: 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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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 , which demonstrated a 100-fold increase in activity over the parent dimethylamiloride in peptide displacement assays.

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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.

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

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Higher sensitivity of NMR spectrometers and novel isotopic labeling schemes have ushered the development of rapid data acquisition methodologies, improving the time resolution with which NMR data can be acquired. For nucleic acids, longitudinal relaxation optimization in conjunction with Ernst angle excitation (SOFAST-HMQC) for imino protons, in addition to rendering rapid pulsing, has been demonstrated to yield significant improvements in sensitivity per unit time. Extending such methodology to other spins offers a viable prospect to measure additional chemical shifts, thereby broadening their utilization for various applications.

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Cellular energy influences all aspects of cellular function. Although cells can adapt to a gradual reduction in energy, acute energy depletion poses a unique challenge. Because acute depletion hampers the transport of new energy sources into the cell, the cell must use endogenous substrates to replenish energy after acute depletion.

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