RESUMO
Notch signaling guides vascular development and function by regulating diverse endothelial cell behaviors, including migration, proliferation, vascular density, endothelial junctions, and polarization in response to flow. Notch proteins form transcriptional activation complexes that regulate endothelial gene expression, but few of the downstream effectors that enable these phenotypic changes have been characterized in endothelial cells, limiting our understanding of vascular Notch activities. Using an unbiased screen of translated mRNA rapidly regulated by Notch signaling, we identified novel in vivo targets of Notch signaling in neonatal mouse brain endothelium, including UNC5B, a member of the netrin family of angiogenic-regulatory receptors. Endothelial Notch signaling rapidly upregulates UNC5B in multiple endothelial cell types. Loss or gain of UNC5B recapitulated specific Notch-regulated phenotypes. UNC5B expression inhibited endothelial migration and proliferation and was required for stabilization of endothelial junctions in response to shear stress. Loss of UNC5B partially or wholly blocked the ability of Notch activation to regulate these endothelial cell behaviors. In the developing mouse retina, endothelial-specific loss of UNC5B led to excessive vascularization, including increased vascular outgrowth, density, and branchpoint count. These data indicate that Notch signaling upregulates UNC5B as an effector protein to control specific endothelial cell behaviors and inhibit angiogenic growth.
Assuntos
Movimento Celular , Proliferação de Células , Células Endoteliais , Receptores de Netrina , Receptores Notch , Retina , Transdução de Sinais , Animais , Receptores de Netrina/metabolismo , Receptores Notch/metabolismo , Camundongos , Células Endoteliais/metabolismo , Retina/metabolismo , Humanos , Vasos Retinianos/metabolismo , Neovascularização FisiológicaRESUMO
Defects in tRNA biogenesis are associated with multiple neurological disorders, yet our understanding of these diseases has been hampered by an inability to determine tRNA expression in individual cell types within a complex tissue. Here, we developed a mouse model in which RNA polymerase III is conditionally epitope tagged in a Cre-dependent manner, allowing us to accurately profile tRNA expression in any cell type in vivo. We investigated tRNA expression in diverse nervous system cell types, revealing dramatic heterogeneity in the expression of tRNA genes between populations. We found that while maintenance of levels of tRNA isoacceptor families is critical for cellular homeostasis, neurons are differentially vulnerable to insults to distinct tRNA isoacceptor families. Cell-type-specific translatome analysis suggests that the balance between tRNA availability and codon demand may underlie such differential resilience. Our work provides a platform for investigating the complexities of mRNA translation and tRNA biology in the brain.
Assuntos
Encéfalo , Homeostase , Neurônios , RNA de Transferência , Animais , RNA de Transferência/genética , RNA de Transferência/metabolismo , Homeostase/fisiologia , Camundongos , Encéfalo/metabolismo , Neurônios/metabolismo , RNA Polimerase III/metabolismo , RNA Polimerase III/genética , Camundongos TransgênicosRESUMO
Transcription factors play vital roles in neuron development; however, little is known about the role of these proteins in maintaining neuronal homeostasis. Here, we show that the transcription factor RREB1 (Ras-responsive element-binding protein 1) is essential for neuron survival in the mammalian brain. A spontaneous mouse mutation causing loss of a nervous system-enriched Rreb1 transcript is associated with progressive loss of cerebellar Purkinje cells and ataxia. Analysis of chromatin immunoprecipitation and sequencing, along with RNA sequencing data revealed dysregulation of RREB1 targets associated with the microtubule cytoskeleton. In agreement with the known role of microtubules in dendritic development, dendritic complexity was disrupted in Rreb1-deficient neurons. Analysis of sequencing data also suggested that RREB1 plays a role in the endomembrane system. Mutant Purkinje cells had fewer numbers of autophagosomes and lysosomes and contained P62- and ubiquitin-positive inclusions. Together, these studies demonstrate that RREB1 functions to maintain the microtubule network and proteostasis in mammalian neurons.
Assuntos
Proteostase , Fatores de Transcrição , Animais , Camundongos , Mamíferos , Microtúbulos , Neurônios , Células de PurkinjeRESUMO
Translational readthrough of UGA stop codons by selenocysteine-specific tRNA (tRNASec) enables the synthesis of selenoproteins. Seryl-tRNA synthetase (SerRS) charges tRNASec with serine, which is modified into selenocysteine and delivered to the ribosome by a designated elongation factor (eEFSec in eukaryotes). Here we found that components of the human selenocysteine incorporation machinery (SerRS, tRNASec, and eEFSec) also increased translational readthrough of non-selenocysteine genes, including VEGFA, to create C-terminally extended isoforms. SerRS recognizes target mRNAs through a stem-loop structure that resembles the variable loop of its cognate tRNAs. This function of SerRS depends on both its enzymatic activity and a vertebrate-specific domain. Through eCLIP-seq, we identified additional SerRS-interacting mRNAs as potential readthrough genes. Moreover, SerRS overexpression was sufficient to reverse premature termination caused by a pathogenic nonsense mutation. Our findings expand the repertoire of selenoprotein biosynthesis machinery and suggest an avenue for therapeutic targeting of nonsense mutations using endogenous factors.
Assuntos
Biossíntese de Proteínas , Serina-tRNA Ligase , Humanos , Códon sem Sentido , Códon de Terminação , RNA Mensageiro/metabolismo , Selenocisteína/genética , Selenocisteína/metabolismo , Selenoproteínas/genética , Serina-tRNA Ligase/genéticaRESUMO
SignificanceUnderstanding and treating neurological disorders are global priorities. Some of these diseases are engendered by mutations that cause defects in the cellular synthesis of transfer RNAs (tRNAs), which function as adapter molecules that translate messenger RNAs into proteins. During tRNA biogenesis, ribonuclease P catalyzes removal of the transcribed sequence upstream of the mature tRNA. Here, we focus on a cytoplasmic tRNAArgUCU that is expressed specifically in neurons and, when harboring a particular point mutation, contributes to neurodegeneration in mice. Our results suggest that this mutation favors stable alternative structures that are not cleaved by mouse ribonuclease P and motivate a paradigm that may help to understand the molecular basis for disease-associated mutations in other tRNAs.
Assuntos
Homeostase , Neurônios/metabolismo , Conformação de Ácido Nucleico , RNA de Transferência/metabolismo , Animais , Pareamento de Bases , Córtex Cerebral/enzimologia , Magnésio/metabolismo , Camundongos , Modelos Moleculares , Mutação Puntual , Processamento de Proteína Pós-Traducional , RNA de Transferência/química , RNA de Transferência/genética , Ribonuclease P/isolamento & purificação , Ribonuclease P/metabolismo , Especificidade por SubstratoRESUMO
Blood-brain barrier (BBB) integrity is critical for proper function of the central nervous system (CNS). Here, we show that the endothelial Unc5B receptor controls BBB integrity by maintaining Wnt/ß-catenin signaling. Inducible endothelial-specific deletion of Unc5B in adult mice leads to BBB leak from brain capillaries that convert to a barrier-incompetent state with reduced Claudin-5 and increased PLVAP expression. Loss of Unc5B decreases BBB Wnt/ß-catenin signaling, and ß-catenin overexpression rescues Unc5B mutant BBB defects. Mechanistically, the Unc5B ligand Netrin-1 enhances Unc5B interaction with the Wnt co-receptor LRP6, induces its phosphorylation and activates Wnt/ß-catenin downstream signaling. Intravenous delivery of antibodies blocking Netrin-1 binding to Unc5B causes a transient BBB breakdown and disruption of Wnt signaling, followed by neurovascular barrier resealing. These data identify Netrin-1-Unc5B signaling as a ligand-receptor pathway that regulates BBB integrity, with implications for CNS diseases.
Assuntos
Barreira Hematoencefálica , Receptores de Netrina , Animais , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Ligantes , Camundongos , Receptores de Netrina/genética , Receptores de Netrina/metabolismo , Netrina-1/genética , Netrina-1/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
Homozygous mutation of the RNA kinase CLP1 (cleavage factor polyribonucleotide kinase subunit 1) causes pontocerebellar hypoplasia type 10 (PCH10), a pediatric neurodegenerative disease. CLP1 is associated with the transfer RNA (tRNA) splicing endonuclease complex and the cleavage and polyadenylation machinery, but its function remains unclear. We generated two mouse models of PCH10: one homozygous for the disease-associated Clp1 mutation, R140H, and one heterozygous for this mutation and a null allele. Both models exhibit loss of lower motor neurons and neurons of the deep cerebellar nuclei. To explore whether Clp1 mutation impacts tRNA splicing, we profiled the products of intron-containing tRNA genes. While mature tRNAs were expressed at normal levels in mutant mice, numerous other products of intron-containing tRNA genes were dysregulated, with pre-tRNAs, introns, and certain tRNA fragments up-regulated, and other fragments down-regulated. However, the spatiotemporal patterns of dysregulation do not correlate with pathogenicity for most altered tRNA products. To elucidate the effect of Clp1 mutation on precursor messenger RNA (pre-mRNA) cleavage, we analyzed poly(A) site (PAS) usage and gene expression in Clp1R140H/- spinal cord. PAS usage was shifted from proximal to distal sites in the mutant mouse, particularly in short and closely spaced genes. Many such genes were also expressed at lower levels in the Clp1R140H/- mouse, possibly as a result of impaired transcript maturation. These findings are consistent with the hypothesis that select genes are particularly dependent upon CLP1 for proper pre-mRNA cleavage, suggesting that impaired mRNA 3' processing may contribute to pathogenesis in PCH10.
Assuntos
Doenças Cerebelares/patologia , Doenças Neurodegenerativas/patologia , Poliadenilação , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/fisiologia , Fatores de Transcrição/fisiologia , Animais , Doenças Cerebelares/genética , Doenças Cerebelares/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA de Transferência/genéticaRESUMO
Translation-dependent quality control pathways such as no-go decay (NGD), non-stop decay (NSD), and nonsense-mediated decay (NMD) govern protein synthesis and proteostasis by resolving non-translating ribosomes and preventing the production of potentially toxic peptides derived from faulty and aberrant mRNAs. However, how translation is altered and the in vivo defects that arise in the absence of these pathways are poorly understood. Here, we show that the NGD/NSD factors Pelo and Hbs1l are critical in mice for cerebellar neurogenesis but expendable for survival of these neurons after development. Analysis of mutant mouse embryonic fibroblasts revealed translational pauses, alteration of signaling pathways, and translational reprogramming. Similar effects on signaling pathways, including mTOR activation, the translatome and mouse cerebellar development were observed upon deletion of the NMD factor Upf2. Our data reveal that these quality control pathways that function to mitigate errors at distinct steps in translation can evoke similar cellular responses.
Assuntos
Proteínas de Ciclo Celular/genética , Cerebelo/crescimento & desenvolvimento , Endonucleases/genética , Proteínas de Ligação ao GTP/genética , Neurogênese/genética , Neurônios/fisiologia , Terminação Traducional da Cadeia Peptídica/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Endonucleases/metabolismo , Feminino , Proteínas de Ligação ao GTP/metabolismo , Masculino , Camundongos , Camundongos KnockoutAssuntos
Licença para Cuidar de Pessoa da Família/legislação & jurisprudência , Internato e Residência/legislação & jurisprudência , Políticas , Radiologia/legislação & jurisprudência , Licença Médica/legislação & jurisprudência , Humanos , Radioterapia (Especialidade)/legislação & jurisprudência , Radiologia Intervencionista/legislação & jurisprudência , Estados UnidosRESUMO
During mRNA translation, tRNAs are charged by aminoacyl-tRNA synthetases and subsequently used by ribosomes. A multi-enzyme aminoacyl-tRNA synthetase complex (MSC) has been proposed to increase protein synthesis efficiency by passing charged tRNAs to ribosomes. An alternative function is that the MSC repurposes specific synthetases that are released from the MSC upon cues for functions independent of translation. To explore this, we generated mammalian cells in which arginyl-tRNA synthetase and/or glutaminyl-tRNA synthetase were absent from the MSC. Protein synthesis, under a variety of stress conditions, was unchanged. Most strikingly, levels of charged tRNAArg and tRNAGln remained unchanged and no ribosome pausing was observed at codons for arginine and glutamine. Thus, increasing or regulating protein synthesis efficiency is not dependent on arginyl-tRNA synthetase and glutaminyl-tRNA synthetase in the MSC. Alternatively, and consistent with previously reported ex-translational roles requiring changes in synthetase cellular localizations, our manipulations of the MSC visibly changed localization.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Biossíntese de Proteínas , RNA de Transferência de Arginina/metabolismo , RNA de Transferência de Glutamina/metabolismo , Ribossomos/metabolismo , Animais , Fibroblastos , Células HEK293 , Humanos , CamundongosRESUMO
Ribosome-associated quality control pathways respond to defects in translational elongation to recycle arrested ribosomes and degrade aberrant polypeptides and mRNAs. Loss of a tRNA gene leads to ribosomal pausing that is resolved by the translational GTPase GTPBP2, and in its absence causes neuron death. Here, we show that loss of the homologous protein GTPBP1 during tRNA deficiency in the mouse brain also leads to codon-specific ribosome pausing and neurodegeneration, suggesting that these non-redundant GTPases function in the same pathway to mitigate ribosome pausing. As observed in Gtpbp2-/- mice (Ishimura et al., 2016), GCN2-mediated activation of the integrated stress response (ISR) was apparent in the Gtpbp1-/- brain. We observed decreased mTORC1 signaling which increased neuronal death, whereas ISR activation was neuroprotective. Our data demonstrate that GTPBP1 functions as an important quality control mechanism during translation elongation and suggest that translational signaling pathways intricately interact to regulate neuronal homeostasis during defective elongation.
Assuntos
Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neurônios/metabolismo , Ribossomos/fisiologia , Animais , Antibacterianos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Transdução de Sinais , Sirolimo/farmacologiaRESUMO
The COVID-19 pandemic has dramatically altered the professional and personal lives of radiologists and radiation oncologists. This article summarizes the 2020 American Association for Women in Radiology (AAWR) Women's Caucus at the American College of Radiology (ACR) Annual Meeting. The caucus focused on the major challenges that women in radiology have faced during the pandemic.
Assuntos
Infecções por Coronavirus , Pandemias , Pneumonia Viral , Radiologia , Betacoronavirus , COVID-19 , Congressos como Assunto , Feminino , Humanos , Radiografia , SARS-CoV-2 , Estados Unidos/epidemiologiaRESUMO
The mammalian genome has hundreds of nuclear-encoded tRNAs, but the contribution of individual tRNA genes to cellular and organismal function remains unknown. Here, we demonstrate that mutations in a neuronally enriched arginine tRNA, n-Tr20, increased seizure threshold and altered synaptic transmission. n-Tr20 expression also modulated seizures caused by an epilepsy-linked mutation in Gabrg2, a gene encoding a GABAA receptor subunit. Loss of n-Tr20 altered translation initiation by activating the integrated stress response and suppressing mTOR signaling, the latter of which may contribute to altered neurotransmission in mutant mice. Deletion of a highly expressed isoleucine tRNA similarly altered these signaling pathways in the brain, suggesting that regulation of translation initiation is a conserved response to tRNA loss. Our data indicate that loss of a single member of a tRNA family results in multiple cellular phenotypes, highlighting the disease-causing potential of tRNA mutations.
Assuntos
Neurônios/metabolismo , RNA de Transferência de Arginina/genética , Convulsões/genética , Transmissão Sináptica/genética , Animais , Eletrochoque/efeitos adversos , Antagonistas de Receptores de GABA-A/efeitos adversos , Camundongos , Pentilenotetrazol/efeitos adversos , Iniciação Traducional da Cadeia Peptídica/genética , RNA de Transferência de Isoleucina/genética , RNA-Seq , Receptores de GABA-A/genética , Convulsões/induzido quimicamente , Convulsões/etiologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
The loss of peroxisomal function has been implicated in many neurodegenerative diseases, yet the underlying molecular mechanisms are poorly understood. In this issue of Neuron, Chung et al. (2020) demonstrate that gain- and loss-of-function mutations in the peroxisomal acyl-CoA oxidase 1 (ACOX1) gene cause neurodegeneration via distinct molecular pathways in glia.
Assuntos
Mutação com Ganho de Função , Metabolismo dos Lipídeos , Acil-CoA Oxidase/genética , Axônios , MutaçãoRESUMO
In the Fig. 3b western blot of this Article, 'Myc-AlaRS' in row one should have been 'Myc-AAD Aars', 'AlaRS' in row two should have been 'Aars' and 'ANKRD16' in row four should have been 'Ankrd16'. In Fig. 4f, 'ANKRD16' and 'ANKRD16(3xR)' should have been 'Ankrd16' and 'Ankrd163xR; and in Fig. 3c the position of the molecular mass markers had shifted. These figures have been corrected online, and see Supplementary Information to the accompanying Amendment for the original figure.
RESUMO
Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aars sti mutant mice results in an increase in the production of serine-mischarged tRNAAla and the degeneration of cerebellar Purkinje cells. Here, using positional cloning, we identified Ankrd16, a gene that acts epistatically with the Aars sti mutation to attenuate neurodegeneration. ANKRD16, a vertebrate-specific protein that contains ankyrin repeats, binds directly to the catalytic domain of AlaRS. Serine that is misactivated by AlaRS is captured by the lysine side chains of ANKRD16, which prevents the charging of serine adenylates to tRNAAla and precludes serine misincorporation in nascent peptides. The deletion of Ankrd16 in the brains of Aarssti/sti mice causes widespread protein aggregation and neuron loss. These results identify an amino-acid-accepting co-regulator of tRNA synthetase editing as a new layer of the machinery that is essential to the prevention of severe pathologies that arise from defects in editing.
Assuntos
Alanina-tRNA Ligase/genética , Alanina-tRNA Ligase/metabolismo , Mutação , Biossíntese de Proteínas , Células de Purkinje/enzimologia , Células de Purkinje/patologia , Alanina/metabolismo , Alanina-tRNA Ligase/química , Animais , Domínio Catalítico , Morte Celular , Feminino , Lisina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Células de Purkinje/metabolismo , Serina/metabolismoRESUMO
Errors during mRNA translation can lead to a reduction in the levels of functional proteins and an increase in deleterious molecules. Advances in next-generation sequencing have led to the discovery of rare genetic disorders, many caused by mutations in genes encoding the mRNA translation machinery, as well as to a better understanding of translational dynamics through ribosome profiling. We discuss here multiple neurological disorders that are linked to errors in tRNA aminoacylation and ribosome decoding. We draw on studies from genetic models, including yeast and mice, to enhance our understanding of the translational defects observed in these diseases. Finally, we emphasize the importance of tRNA, their associated enzymes, and the inextricable link between accuracy and efficiency in the maintenance of translational fidelity.
Assuntos
Mutação , Doenças do Sistema Nervoso/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Animais , Humanos , Modelos Genéticos , Saccharomyces cerevisiae/genética , Aminoacilação de RNA de Transferência/genéticaRESUMO
Dynamic regulation of mRNA translation initiation and elongation is essential for the survival and function of neural cells. Global reductions in translation initiation resulting from mutations in the translational machinery or inappropriate activation of the integrated stress response may contribute to pathogenesis in a subset of neurodegenerative disorders. Aberrant proteins generated by non-canonical translation initiation may be a factor in the neuron death observed in the nucleotide repeat expansion diseases. Dysfunction of central components of the elongation machinery, such as the tRNAs and their associated enzymes, can cause translational infidelity and ribosome stalling, resulting in neurodegeneration. Taken together, dysregulation of mRNA translation is emerging as a unifying mechanism underlying the pathogenesis of many neurodegenerative disorders.