Kevin Rhine

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• Rhine, K., Li, R., Kopalle, H. M., Rothamel, K., Ge, X., Epstein, E., Mizrahi, O., Madrigal, A. A., Her, H. L., Gomberg, T. A., Hermann, A., Schwartz, J. L., Daniels, A. J., Manor, U., Ravits, J., Signer, R. A., Bennett, E. J., & Yeo, G. W. (2025). Neuronal aging causes mislocalization of splicing proteins and unchecked cellular stress. Nature Neuroscience, 28(Issue 6). doi:10.1038/s41593-025-01952-z
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  Aging is one of the most prominent risk factors for neurodegeneration, yet the molecular mechanisms underlying the deterioration of old neurons are mostly unknown. To efficiently study neurodegeneration in the context of aging, we transdifferentiated primary human fibroblasts from aged healthy donors directly into neurons, which retained their aging hallmarks, and we verified key findings in aged human and mouse brain tissue. Here we show that aged neurons are broadly depleted of RNA-binding proteins, especially spliceosome components. Intriguingly, splicing proteins—like the dementia- and ALS-associated protein TDP-43—mislocalize to the cytoplasm in aged neurons, which leads to widespread alternative splicing. Cytoplasmic spliceosome components are typically recruited to stress granules, but aged neurons suffer from chronic cellular stress that prevents this sequestration. We link chronic stress to the malfunctioning ubiquitylation machinery, poor HSP90α chaperone activity and the failure to respond to new stress events. Together, our data demonstrate that aging-linked deterioration of RNA biology is a key driver of poor resiliency in aged neurons.
• Liang, Q., Yu, T., Kofman, E., Jagannatha, P., Rhine, K., Yee, B. A., Corbett, K. D., & Yeo, G. W. (2024). High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissues. Nature Communications, 15(Issue 1). doi:10.1038/s41467-024-50363-4
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  RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue and accurately identifies the canonical binding motifs of RBFOX2 (UGCAUG) and TDP-43 (UGUGUG) in native cellular environments. Applicable to any RBP with available primary antibodies, INSCRIBE enables sensitive capture of RBP-RNA interactions from ultra-low input equivalent to ~5 cells. The robust, versatile, and sensitive INSCRIBE workflow is particularly beneficial for precious tissues such as clinical samples, empowering the exploration of genuine RBP-RNA interactions in RNA-related disease contexts.
• Street, L. A., Rothamel, K. L., Brannan, K. W., Jin, W., Bokor, B. J., Dong, K., Rhine, K., Madrigal, A., Al-Azzam, N., Kim, J. K., Ma, Y., Gorhe, D., Abdou, A., Wolin, E., Mizrahi, O., Ahdout, J., Mujumdar, M., Doron-Mandel, E., Jovanovic, M., & Yeo, G. W. (2024). Large-scale map of RNA-binding protein interactomes across the mRNA life cycle. Molecular Cell, 84(Issue 19). doi:10.1016/j.molcel.2024.08.030
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  mRNAs interact with RNA-binding proteins (RBPs) throughout their processing and maturation. While efforts have assigned RBPs to RNA substrates, less exploration has leveraged protein-protein interactions (PPIs) to study proteins in mRNA life-cycle stages. We generated an RNA-aware, RBP-centric PPI map across the mRNA life cycle in human cells by immunopurification-mass spectrometry (IP-MS) of ∼100 endogenous RBPs with and without RNase, augmented by size exclusion chromatography-mass spectrometry (SEC-MS). We identify 8,742 known and 20,802 unreported interactions between 1,125 proteins and determine that 73% of the IP-MS-identified interactions are RNA regulated. Our interactome links many proteins, some with unknown functions, to specific mRNA life-cycle stages, with nearly half associated with multiple stages. We demonstrate the value of this resource by characterizing the splicing and export functions of enhancer of rudimentary homolog (ERH), and by showing that small nuclear ribonucleoprotein U5 subunit 200 (SNRNP200) interacts with stress granule proteins and binds cytoplasmic RNA differently during stress.
• Fare, C. M., Rhine, K., Lam, A., Myong, S., & Shorter, J. (2023). A minimal construct of nuclear-import receptor Karyopherin-β2 defines the regions critical for chaperone and disaggregation activity. Journal of Biological Chemistry, 299(Issue 2). doi:10.1016/j.jbc.2022.102806
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  Karyopherin-β2 (Kapβ2) is a nuclear-import receptor that recognizes proline-tyrosine nuclear localization signals of diverse cytoplasmic cargo for transport to the nucleus. Kapβ2 cargo includes several disease-linked RNA-binding proteins with prion-like domains, such as FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2. These RNA-binding proteins with prion-like domains are linked via pathology and genetics to debilitating degenerative disorders, including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Remarkably, Kapβ2 prevents and reverses aberrant phase transitions of these cargoes, which is cytoprotective. However, the molecular determinants of Kapβ2 that enable these activities remain poorly understood, particularly from the standpoint of nuclear-import receptor architecture. Kapβ2 is a super-helical protein comprised of 20 HEAT repeats. Here, we design truncated variants of Kapβ2 and assess their ability to antagonize FUS aggregation and toxicity in yeast and FUS condensation at the pure protein level and in human cells. We find that HEAT repeats 8 to 20 of Kapβ2 recapitulate all salient features of Kapβ2 activity. By contrast, Kapβ2 truncations lacking even a single cargo-binding HEAT repeat display reduced activity. Thus, we define a minimal Kapβ2 construct for delivery in adeno-associated viruses as a potential therapeutic for amyotrophic lateral sclerosis/frontotemporal dementia, multisystem proteinopathy, and related disorders.
• Rhine, K., & Myong, S. (2022). Single molecule probing of disordered RNA binding proteins. STAR Protocols, 3(Issue 1). doi:10.1016/j.xpro.2022.101131
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  Liquid-liquid phase separation of intrinsically disordered proteins is known to underlie diverse pathologies such as neurodegeneration, cancer, and aging. The nucleation step of condensate formation is of critical importance for understanding how healthy and disease-associated condensates differ. Here, we describe four orthogonal single-molecule techniques that enable molecular tracking of the RNA-protein interaction, RNA-induced oligomerization, and kinetics of nucleation. These approaches allow researchers to directly interrogate the initial steps of liquid-liquid phase separation. For complete details on the use and execution of this profile, please refer to Niaki et al. (2020), Rhine et al. (2020), and Rhine et al. (2022).
• Rhine, K., Dasovich, M., Yoniles, J., Badiee, M., Skanchy, S., Ganser, L. R., Ge, Y., Fare, C. M., Shorter, J., Leung, A. K., & Myong, S. (2022). Poly(ADP-ribose) drives condensation of FUS via a transient interaction. Molecular Cell, 82(Issue 5). doi:10.1016/j.molcel.2022.01.018
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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. 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.
• Rhine, K., Skanchy, S., & Myong, S. (2022). Single-molecule and ensemble methods to probe RNP nucleation and condensate properties. Methods, 197(Issue). doi:10.1016/j.ymeth.2021.02.012
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  Biomolecular condensates often consist of intrinsically disordered protein and RNA molecules, which together promote the formation of membraneless organelles in cells. The nucleation, condensation, and maturation of condensates is a critical yet poorly understood process. Here, we present single-molecule and accompanying ensemble methods to quantify these processes more comprehensively. In particular, we focus on how to properly design and execute a single-molecule nucleation assay, in which we detect signals arising from individual units of fluorescently labeled RNA-binding proteins associating with an RNA substrate. The analysis of this data allows one to determine the kinetics involved with each step of nucleation. Complemented with meso-scale techniques that measure the biophysical properties of ribonucleoprotein condensates, the methods described herein are powerful tools that can be adopted for studying any protein-RNA interactions that promote phase separation.
• Hui, M. H., Rhine, K., & Tolan, D. R. (2021). Actin filament- and Wiskott-Aldrich syndrome protein-binding sites on fructose-1,6-bisphosphate aldolase are functionally distinct from the active site. Cytoskeleton, 78(Issue 4). doi:10.1002/cm.21646
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  The glycolytic enzyme fructose 1,6-(bis)phosphate aldolase (aldolase) is not only required for efficient utilization of glucose and fructose, but also for cytoskeletal functions like cytokinesis and cell motility. These differing roles are mediated by distinct and discrete binding interactions with aldolase's many binding partners, including actin filaments, Wiskott-Aldrich Syndrome protein (WASP), and Sorting Nexin 9 (SNX9). How these interactions are coordinated on the aldolase homotetramer of 160 kDa is unclear. In this study, the catalytic activity of wild-type aldolase is measured in the presence of actin filaments, and a WASP-derived peptide that binds to aldolase, or both. No appreciable changes in kcat or Km values are seen. Then, aldolase variants with substitutions targeting the tryptophan-binding pocket for WASP and SNX9 are created and perturbation of actin filament-, WASP peptide-, and SNX9 peptide-binding are assessed. Those that negatively impacted binding did not show an impact on aldolase catalysis. These results suggest that aldolase can engage in catalysis while simultaneously interacting with cytoskeletal machinery.
• Niaki, A. G., Sarkar, J., Cai, X., Rhine, K., Vidaurre, V., Guy, B., Hurst, M., Lee, J. C., Koh, H. R., Guo, L., Fare, C. M., Shorter, J., & Myong, S. (2020). Loss of Dynamic RNA Interaction and Aberrant Phase Separation Induced by Two Distinct Types of ALS/FTD-Linked FUS Mutations. Molecular Cell, 77(Issue 1). doi:10.1016/j.molcel.2019.09.022
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  FUS aggregation is pathogenic hallmark of incurable neurodegenerative diseases, including ALS and FTD. Niaki et al. demonstrate that two classes of ALS/FTD-linked mutations display distinct molecular phenotypes. Arginine mutations lead to defective RNA binding, whereas glycine mutations induce quick loss of fluidity, underpinning potentially disparate pathogenic pathways of disease-linked FUS mutants. FUS is a nuclear RNA-binding protein, and its cytoplasmic aggregation is a pathogenic signature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). It remains unknown how the FUS-RNA interactions contribute to phase separation and whether its phase behavior is affected by ALS-linked mutations. Here we demonstrate that wild-type FUS binds single-stranded RNA stoichiometrically in a length-dependent manner and that multimers induce highly dynamic interactions with RNA, giving rise to small and fluid condensates. In contrast, mutations in arginine display a severely altered conformation, static binding to RNA, and formation of large condensates, signifying the role of arginine in driving proper RNA interaction. Glycine mutations undergo rapid loss of fluidity, emphasizing the role of glycine in promoting fluidity. Strikingly, the nuclear import receptor Karyopherin-β2 reverses the mutant defects and recovers the wild-type FUS behavior. We reveal two distinct mechanisms underpinning potentially disparate pathogenic pathways of ALS-linked FUS mutants.
• Rhine, K., Makurath, M. A., Liu, J., Skanchy, S., Lopez, C., Catalan, K. F., Ma, Y., Fare, C. M., Shorter, J., Ha, T., Chemla, Y. R., & Myong, S. (2020). Erratum: ALS/FTLD-Linked Mutations in FUS Glycine Residues Cause Accelerated Gelation and Reduced Interactions with Wild-Type FUS (Molecular Cell (2020) 80(4) (666–681.e8), (S1097276520307218), (10.1016/j.molcel.2020.10.014)). Molecular Cell, 80(Issue 6). doi:10.1016/j.molcel.2020.11.031
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  (Molecular Cell 80, 666–681.e1–e8; November 19, 2020) In the originally published version of this article, two co-authors, Charlotte M. Fare and James Shorter, were mistakenly excluded from the author list. The article with a corrected author list and author contributions section now appears online. The authors apologize for this error.
• Rhine, K., Makurath, M. A., Liu, J., Skanchy, S., Lopez, C., Catalan, K. F., Ma, Y., Ha, T., Chemla, Y. R., & Myong, S. (2020). ALS/FTLD-Linked Mutations in FUS Glycine Residues Cause Accelerated Gelation and Reduced Interactions with Wild-Type FUS. Molecular Cell, 80(Issue 4). doi:10.1016/j.molcel.2020.10.014
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  The RNA-binding protein fused in sarcoma (FUS) can form pathogenic inclusions in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). Over 70 mutations in Fus are linked to ALS/FTLD. In patients, all Fus mutations are heterozygous, indicating that the mutant drives disease progression despite the presence of wild-type (WT) FUS. Here, we demonstrate that ALS/FTLD-linked FUS mutations in glycine (G) strikingly drive formation of droplets that do not readily interact with WT FUS, whereas arginine (R) mutants form mixed condensates with WT FUS. Remarkably, interactions between WT and G mutants are disfavored at the earliest stages of FUS nucleation. In contrast, R mutants physically interact with the WT FUS such that WT FUS recovers the mutant defects by reducing droplet size and increasing dynamic interactions with RNA. This result suggests disparate molecular mechanisms underlying ALS/FTLD pathogenesis and differing recovery potential depending on the type of mutation. FUS is an RNA-binding protein with over 70 mutations linked to the incurable neurodegenerative diseases ALS and FTLD. Rhine et al. demonstrate that FUS mutations in glycine do not associate with the wild type at all levels, while the mutations in arginine interact with wild-type FUS and thereby recover the mutant defect.