RESUMEN
Dipeptide repeat proteins (DPRs) are aberrant protein species found in C9orf72-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two neurodegenerative diseases characterized by the cytoplasmic mislocalization and aggregation of RNA-binding proteins (RBPs). In particular, arginine (R)-rich DPRs (poly-GR and poly-PR) have been suggested to promiscuously interact with multiple cellular proteins and thereby exert high cytotoxicity. Components of the protein arginine methylation machinery have been identified as modulators of DPR toxicity and/or potential cellular interactors of R-rich DPRs; however, the molecular details and consequences of such an interaction are currently not well understood. Here, we demonstrate that several members of the family of protein arginine methyltransferases (PRMTs) can directly interact with R-rich DPRs in vitro and in the cytosol. In vitro, R-rich DPRs reduce solubility and promote phase separation of PRMT1, the main enzyme responsible for asymmetric arginine-dimethylation (ADMA) in mammalian cells, in a concentration- and length-dependent manner. Moreover, we demonstrate that poly-GR interferes more efficiently than poly-PR with PRMT1-mediated arginine methylation of RBPs such as hnRNPA3. We additionally show by two alternative approaches that poly-GR itself is a substrate for PRMT1-mediated arginine dimethylation. We propose that poly-GR may act as a direct competitor for arginine methylation of cellular PRMT1 targets, such as disease-linked RBPs.
Asunto(s)
Arginina , Proteína-Arginina N-Metiltransferasas , Proteínas de Unión al ARN , Proteínas Represoras , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Humanos , Arginina/metabolismo , Metilación , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/genética , Proteína C9orf72/metabolismo , Proteína C9orf72/genética , Células HEK293RESUMEN
Research into how the young and old differ, and which biomarkers reflect the diverse biological processes underlying ageing, is a current and fast-growing field. Biological clocks provide a means to evaluate whether a molecule, cell, tissue or even an entire organism is old or young. Here we summarize established and emerging molecular clocks as timepieces. We emphasize that intrinsically disordered proteins (IDPs) tend to transform into a ß-sheet-rich aggregated state and accumulate in non-dividing or slowly dividing cells as they age. We hypothesize that understanding these protein-based molecular ageing mechanisms might provide a conceptual pathway to determining a cell's health age by probing the aggregation state of IDPs, which we term the IDP clock.
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Envejecimiento , Relojes Biológicos , Proteínas Intrínsecamente Desordenadas , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Humanos , Envejecimiento/metabolismo , Envejecimiento/genética , Animales , Senescencia CelularRESUMEN
Mutations in the human PURA gene cause the neurodevelopmental PURA syndrome. In contrast to several other monogenetic disorders, almost all reported mutations in this nucleic acid-binding protein result in the full disease penetrance. In this study, we observed that patient mutations across PURA impair its previously reported co-localization with processing bodies. These mutations either destroyed the folding integrity, RNA binding, or dimerization of PURA. We also solved the crystal structures of the N- and C-terminal PUR domains of human PURA and combined them with molecular dynamics simulations and nuclear magnetic resonance measurements. The observed unusually high dynamics and structural promiscuity of PURA indicated that this protein is particularly susceptible to mutations impairing its structural integrity. It offers an explanation why even conservative mutations across PURA result in the full penetrance of symptoms in patients with PURA syndrome.
PURA syndrome is a neurodevelopmental disorder that affects about 650 patients worldwide, resulting in a range of symptoms including neurodevelopmental delays, intellectual disability, muscle weakness, seizures, and eating difficulties. The condition is caused by a mutated gene that codes for a protein called PURA. PURA binds RNA the molecule that carries genetic information so it can be translated into proteins and has roles in regulating the production of new proteins. Contrary to other conditions that result from mutations in a single gene, PURA syndrome patients show 'high penetrance', meaning almost every reported mutation in the gene leads to symptoms. Proske, Janowski et al. wanted to understand the molecular basis for this high penetrance. To find out more, the researchers first examined how patient mutations affected the location of the PURA in the cell, using human cells grown in the laboratory. Normally, PURA travels to P-bodies, which are groupings of RNA and proteins involved in regulating which genes get translated into proteins. The researchers found that in cells carrying PURA syndrome mutations, PURA failed to move adequately to P-bodies. To find out how this 'mislocalization' might happen, Proske, Janowski et al. tested how different mutations affected the three-dimensional folding of PURA. These analyses showed that the mutations impair the protein's folding and thereby disrupt PURA's ability to bind RNA, which may explain why mutant PURA cannot localize correctly. Proske, Janowski et al. describe the molecular abnormalities of PURA underlying this disorder and show how molecular analysis of patient mutations can reveal the mechanisms of a disease at the cell level. The results show that the impact of mutations on the structural integrity of the protein, which affects its ability to bind RNA, are likely key to the symptoms of the syndrome. Additionally, their approach used establishes a way to predict and test mutations that will cause PURA syndrome. This may help to develop diagnostic tools for this condition.
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Trastornos del Neurodesarrollo , Cuerpos de Procesamiento , Humanos , Trastornos del Neurodesarrollo/metabolismo , Trastornos del Neurodesarrollo/patología , Cuerpos de Procesamiento/metabolismo , Cuerpos de Procesamiento/patología , Gránulos de Estrés/metabolismo , Cristalografía por Rayos X , Dimerización , Dominios Proteicos , Dicroismo Circular , Proteínas Recombinantes , Pliegue de Proteína , Penetrancia , Sustitución de Aminoácidos , Mutación Puntual , Células HeLaRESUMEN
TDP-43-positive inclusions in neurons are a hallmark of several neurodegenerative diseases including familial amyotrophic lateral sclerosis (fALS) caused by pathogenic TARDBP variants as well as more common non-Mendelian sporadic ALS (sALS). Here we report a G376V-TDP-43 missense variant in the C-terminal prion-like domain of the protein in two French families affected by an autosomal dominant myopathy but not fulfilling diagnostic criteria for ALS. Patients from both families presented with progressive weakness and atrophy of distal muscles, starting in their 5th-7th decade. Muscle biopsies revealed a degenerative myopathy characterized by accumulation of rimmed (autophagic) vacuoles, disruption of sarcomere integrity and severe myofibrillar disorganization. The G376â V variant altered a highly conserved amino acid residue and was absent in databases on human genome variation. Variant pathogenicity was supported by in silico analyses and functional studies. The G376â V mutant increased the formation of cytoplasmic TDP-43 condensates in cell culture models, promoted assembly into high molecular weight oligomers and aggregates in vitro, and altered morphology of TDP-43 condensates arising from phase separation. Moreover, the variant led to the formation of cytoplasmic TDP-43 condensates in patient-derived myoblasts and induced abnormal mRNA splicing in patient muscle tissue. The identification of individuals with TDP-43-related myopathy but not ALS implies that TARDBP missense variants may have more pleiotropic effects than previously anticipated and support a primary role for TDP-43 in skeletal muscle pathophysiology. We propose to include TARDBP screening in the genetic work-up of patients with late-onset distal myopathy. Further research is warranted to examine the precise pathogenic mechanisms of TARDBP variants causing either a neurodegenerative or myopathic phenotype.
RESUMEN
Cytoplasmic aggregation and concomitant nuclear clearance of the RNA-binding protein TDP-43 are found in ~ 90% of cases of amyotrophic lateral sclerosis and ~ 45% of patients living with frontotemporal lobar degeneration, but no disease-modifying therapy is available. Antibody therapy targeting other aggregating proteins associated with neurodegenerative disorders has shown beneficial effects in animal models and clinical trials. The most effective epitopes for safe antibody therapy targeting TDP-43 are unknown. Here, we identified safe and effective epitopes in TDP-43 for active and potential future passive immunotherapy. We prescreened 15 peptide antigens covering all regions of TDP-43 to identify the most immunogenic epitopes and to raise novel monoclonal antibodies in wild-type mice. Most peptides induced a considerable antibody response and no antigen triggered obvious side effects. Thus, we immunized mice with rapidly progressing TDP-43 proteinopathy ("rNLS8" model) with the nine most immunogenic peptides in five pools prior to TDP-43ΔNLS transgene induction. Strikingly, combined administration of two N-terminal peptides induced genetic background-specific sudden lethality in several mice and was therefore discontinued. Despite a strong antibody response, no TDP-43 peptide prevented the rapid body weight loss or reduced phospho-TDP-43 levels as well as the profound astrogliosis and microgliosis in rNLS8 mice. However, immunization with a C-terminal peptide containing the disease-associated phospho-serines 409/410 significantly lowered serum neurofilament light chain levels, indicative of reduced neuroaxonal damage. Transcriptomic profiling showed a pronounced neuroinflammatory signature (IL-1ß, TNF-α, NfκB) in rNLS8 mice and suggested modest benefits of immunization targeting the glycine-rich region. Several novel monoclonal antibodies targeting the glycine-rich domain potently reduced phase separation and aggregation of TDP-43 in vitro and prevented cellular uptake of preformed aggregates. Our unbiased screen suggests that targeting the RRM2 domain and the C-terminal region of TDP-43 by active or passive immunization may be beneficial in TDP-43 proteinopathies by inhibiting cardinal processes of disease progression.
Asunto(s)
Anticuerpos Monoclonales , Filamentos Intermedios , Animales , Ratones , Epítopos , Inmunización , FN-kappa BRESUMEN
In the last years, RNA-binding proteins (RBPs) have been highlighted for their capacity to undergo liquid-liquid phase separation (LLPS). Aberrant phase transitions of RBPs from a liquid to a solid state are believed to underlie the formation of pathological RBP aggregates in several neurodegenerative diseases. Both in the physiological and the disease state, RBPs are often decorated with diverse posttranslational modifications (PTMs) that can influence the phase separation behavior, the physiological function, and the pathological behavior of the RBP. Here we describe two simple methods, sedimentation assays in vitro and in cells, that allow the analysis of RBP solubility as a measure of RBP phase separation in the absence or presence of a certain PTM.
Asunto(s)
Enfermedades Neurodegenerativas , Proteínas de Unión al ARN , Gránulos Citoplasmáticos/metabolismo , Humanos , Enfermedades Neurodegenerativas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN/metabolismoRESUMEN
Decreasing the activation of pathology-activated microglia is crucial to prevent chronic inflammation and tissue scarring. In this study, we used a stab wound injury model in zebrafish and identified an injury-induced microglial state characterized by the accumulation of lipid droplets and TAR DNA-binding protein of 43 kDa (TDP-43)+ condensates. Granulin-mediated clearance of both lipid droplets and TDP-43+ condensates was necessary and sufficient to promote the return of microglia back to the basal state and achieve scarless regeneration. Moreover, in postmortem cortical brain tissues from patients with traumatic brain injury, the extent of microglial activation correlated with the accumulation of lipid droplets and TDP-43+ condensates. Together, our results reveal a mechanism required for restoring microglia to a nonactivated state after injury, which has potential for new therapeutic applications in humans.
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Lesiones Traumáticas del Encéfalo , Microglía , Humanos , Animales , Gotas Lipídicas , Pez Cebra , Proteínas de Unión al ADN , RegeneraciónRESUMEN
Ubiquilin-2 (UBQLN2) is a ubiquitin-binding protein that shuttles ubiquitinated proteins to proteasomal and autophagic degradation. UBQLN2 mutations are genetically linked to the neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). However, it remains elusive how UBQLN2 mutations cause ALS/FTD. Here, we systematically examined proteomic and transcriptomic changes in patient-derived lymphoblasts and CRISPR/Cas9-engineered HeLa cells carrying ALS/FTD UBQLN2 mutations. This analysis revealed a strong up-regulation of the microtubule-associated protein 1B (MAP1B) which was also observed in UBQLN2 knockout cells and primary rodent neurons depleted of UBQLN2, suggesting that a UBQLN2 loss-of-function mechanism is responsible for the elevated MAP1B levels. Consistent with MAP1B's role in microtubule binding, we detected an increase in total and acetylated tubulin. Furthermore, we uncovered that UBQLN2 mutations result in decreased phosphorylation of MAP1B and of the ALS/FTD-linked fused in sarcoma (FUS) protein at S439 which is critical for regulating FUS-RNA binding and MAP1B protein abundance. Together, our findings point to a deregulated UBQLN2-FUS-MAP1B axis that may link protein homeostasis, RNA metabolism, and cytoskeleton dynamics, three molecular pathomechanisms of ALS/FTD.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Esclerosis Amiotrófica Lateral , Proteínas Relacionadas con la Autofagia , Demencia Frontotemporal , Proteínas Asociadas a Microtúbulos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteómica , ARN/genética , ARN/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
To elucidate the mechanism driving selective autophagy of protein aggregates, or "aggrephagy," Ma et al. (2022) identify chaperonin TRiC subunit CCT2 as a receptor that specifically promotes the clearance of solid aggregates, but not liquid-like condensates, in a ubiquitin-independent manner.
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Residuos Sólidos , Ubiquitina , Autofagia , Proteínas Portadoras/metabolismo , Agregado de Proteínas , Ubiquitina/metabolismoRESUMEN
In the recent years, defective nuclear import has emerged as an important pathomechanism of neurodegenerative diseases, particularly in amyotrophic lateral sclerosis (ALS). Here, specific nuclear RNA binding proteins (RBPs) mislocalize and aggregate in the cytoplasm of neurons and glial cells in degenerating brain regions. Bona fide transport assays that measure nuclear import in a quantitative manner allow one to distinguish whether disease-linked RBP mutations that cause cytosolic RBP mislocalization directly result in reduced nuclear import or cause increased cytoplasmic localization of the RBP through other mechanisms. Here we describe the quantitative analysis of nuclear import rates of RBPs using a hormone-inducible system by live cell imaging.
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Mediciones Luminiscentes , Enfermedades Neurodegenerativas , Proteínas de Unión al ARN , Transporte Activo de Núcleo Celular/fisiología , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Citoplasma/metabolismo , Proteínas Fluorescentes Verdes , Hormonas/metabolismo , Humanos , Mediciones Luminiscentes/métodos , Microscopía Fluorescente/métodos , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Post-translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis and frontotemporal dementia, the RNA-binding protein TAR DNA-binding protein (TDP-43), is hyperphosphorylated in disease on several C-terminal serine residues, a process generally believed to promote TDP-43 aggregation. Here, we however find that Casein kinase 1δ-mediated TDP-43 hyperphosphorylation or C-terminal phosphomimetic mutations reduce TDP-43 phase separation and aggregation, and instead render TDP-43 condensates more liquid-like and dynamic. Multi-scale molecular dynamics simulations reveal reduced homotypic interactions of TDP-43 low-complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP-43, but suppress accumulation of TDP-43 in membrane-less organelles and promote its solubility in neurons. We speculate that TDP-43 hyperphosphorylation may be a protective cellular response to counteract TDP-43 aggregation.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Agregado de Proteínas , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
RNA-binding proteins (RBPs) are critical players in RNA expression and metabolism, thus, the proper regulation of this class of proteins is critical for cellular health. Regulation of RBPs often occurs through post-translational modifications (PTMs), which allow the cell to quickly and efficiently respond to cellular and environmental stimuli. PTMs have recently emerged as important regulators of RBPs implicated in neurodegenerative disorders, in particular amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we summarize how disease-associated PTMs influence the biophysical properties, molecular interactions, subcellular localization, and function of ALS/FTD-linked RBPs, such as FUS and TDP-43. We will discuss how PTMs are believed to play pathological, protective, or ambiguous roles in these neurodegenerative disorders.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Humanos , Procesamiento Proteico-Postraduccional , Proteína FUS de Unión a ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Arginine-glycine(-glycine) (RG/RGG) regions are highly abundant in RNA-binding proteins and involved in numerous physiological processes. Aberrant liquid-liquid phase separation (LLPS) and stress granule (SGs) association of RG/RGG regions in the cytoplasm have been implicated in several neurodegenerative disorders. LLPS and SG association of these proteins is regulated by the interaction with nuclear import receptors, such as transportin-1 (TNPO1), and by post-translational arginine methylation. Strikingly, many RG/RGG proteins harbour potential phosphorylation sites within or close to their arginine methylated regions, indicating a regulatory role. Here, we studied the role of phosphorylation within RG/RGG regions on arginine methylation, TNPO1-binding and LLPS using the cold-inducible RNA-binding protein (CIRBP) as a paradigm. We show that the RG/RGG region of CIRBP is in vitro phosphorylated by serine-arginine protein kinase 1 (SRPK1), and discovered two novel phosphorylation sites in CIRBP. SRPK1-mediated phosphorylation of the CIRBP RG/RGG region impairs LLPS and binding to TNPO1 in vitro and interferes with SG association in cells. Furthermore, we uncovered that arginine methylation of the CIRBP RG/RGG region regulates in vitro phosphorylation by SRPK1. In conclusion, our findings indicate that LLPS and TNPO1-mediated chaperoning of RG/RGG proteins is regulated through an intricate interplay of post-translational modifications.
RESUMEN
Liquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. The protein Fused in Sarcoma (FUS) undergoes LLPS and mutations in FUS have been causally linked to the motor neuron disease Amyotrophic Lateral Sclerosis (ALS-FUS). LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. However, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. Hence, we developed a method allowing for the purification of LLPS FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome, depending on its biophysical state. While non-LLPS FUS interacts mainly with factors involved in pre-mRNA processing, LLPS FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, also mitochondrial factors are strongly enriched with LLPS FUS, providing a potential explanation for the observed changes in mitochondrial gene expression in mouse models of ALS-FUS. In summary, we present a methodology to investigate the interactomes of phase separating proteins and provide evidence that LLPS shapes the FUS interactome with implications for function and disease.
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Proteína FUS de Unión a ARN/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Citoplasma/metabolismo , Gránulos Citoplasmáticos/metabolismo , Células HEK293 , Células HeLa , Humanos , Mapeo de Interacción de Proteínas , ARN Mensajero/metabolismo , ARN Nuclear Pequeño/metabolismo , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/aislamiento & purificaciónRESUMEN
Fused in sarcoma (FUS) is a predominantly nuclear RNA-binding protein with key functions in RNA processing and DNA damage repair. Defects in nuclear import of FUS have been linked to severe neurodegenerative diseases; hence, it is of great interest to understand this process and how it is dysregulated in disease. Transportin-1 (TNPO1) and the closely related transportin-2 have been identified as major nuclear import receptors of FUS. They bind to the C-terminal nuclear localization signal of FUS and mediate the protein's nuclear import and at the same time also suppress aberrant phase transitions of FUS in the cytoplasm. Whether FUS can utilize other nuclear transport receptors for the purpose of import and chaperoning has not been examined so far. Here, we show that FUS directly binds to different import receptors in vitro. FUS formed stable complexes not only with TNPO1 but also with transportin-3, importin ß, importin 7, or the importin ß/7 heterodimer. Binding of these alternative import receptors required arginine residues within FUS-RG/RGG motifs and was weakened by arginine methylation. Interaction with these importins suppressed FUS phase separation and reduced its sequestration into stress granules. In a permeabilized cell system, we further showed that transportin-3 had the capacity to import FUS into the nucleus, albeit with lower efficiency than TNPO1. Our data suggest that aggregation-prone RNA-binding proteins such as FUS may utilize a network of importins for chaperoning and import, similar to histones and ribosomal proteins.
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Núcleo Celular/metabolismo , Carioferinas/metabolismo , Chaperonas Moleculares/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , beta Carioferinas/metabolismo , Núcleo Celular/genética , Células HeLa , Humanos , Carioferinas/genética , Chaperonas Moleculares/genética , Señales de Localización Nuclear , Unión Proteica , Proteína FUS de Unión a ARN/genética , Receptores Citoplasmáticos y Nucleares/genética , beta Carioferinas/genéticaRESUMEN
Nuclear import receptors, also called importins, mediate nuclear import of proteins and chaperone aggregation-prone cargoes (e.g., neurodegeneration-linked RNA-binding proteins [RBPs]) in the cytoplasm. Importins were identified as modulators of cellular toxicity elicited by arginine-rich dipeptide repeat proteins (DPRs), an aberrant protein species found in C9orf72-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Mechanistically, the link between importins and arginine-rich DPRs remains unclear. Here, we show that arginine-rich DPRs (poly-GR and poly-PR) bind directly to multiple importins and, in excess, promote their insolubility and condensation. In cells, poly-GR impairs Impα/ß-mediated nuclear import, including import of TDP-43, an RBP that aggregates in C9orf72-ALS/FTD patients. Arginine-rich DPRs promote phase separation and insolubility of TDP-43 in vitro and in cells, and this pathological interaction is suppressed by elevating importin concentrations. Our findings suggest that importins can decrease toxicity of arginine-rich DPRs by suppressing their pathological interactions.
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Arginina/metabolismo , Dipéptidos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , HumanosRESUMEN
The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)-rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.