RESUMO
Fused in sarcoma (FUS) is a pathogenic RNA-binding protein in amyotrophic lateral sclerosis (ALS). We previously reported that FUS stabilizes Synaptic Ras-GTPase activating protein 1 (Syngap1) mRNA at its 3' untranslated region (UTR) and maintains spine maturation. To elucidate the pathologic roles of this mechanism in ALS patients, we identified the SYNGAP1 3'UTR variant rs149438267 in seven (four males and three females) out of 807 ALS patients at the FUS binding site from a multicenter cohort in Japan. Human-induced pluripotent stem cell (hiPSC)-derived motor neurons with the SYNGAP1 variant showed aberrant splicing, increased isoform α1 levels, and decreased isoform γ levels, which caused dendritic spine loss. Moreover, the SYNGAP1 variant excessively recruited FUS and heterogeneous nuclear ribonucleoprotein K (HNRNPK), and antisense oligonucleotides (ASOs) blocking HNRNPK altered aberrant splicing and ameliorated dendritic spine loss. These data suggest that excessive recruitment of RNA-binding proteins, especially HNRNPK, as well as changes in SYNGAP1 isoforms, are crucial for spine formation in motor neurons.SIGNIFICANCE STATEMENT It is not yet known which RNAs cause the pathogenesis of amyotrophic lateral sclerosis (ALS). We previously reported that Fused in sarcoma (FUS), a pathogenic RNA-binding protein in ALS, stabilizes synaptic Ras-GTPase activating protein 1 (Syngap1) mRNA at its 3' untranslated region (UTR) and maintains dendritic spine maturation. To elucidate whether this mechanism is crucial for ALS, we identified the SYNGAP1 3'UTR variant rs149438267 at the FUS binding site. Human-induced pluripotent stem cell (hiPSC)-derived motor neurons with the SYNGAP1 variant showed aberrant splicing, which caused dendritic spine loss along with excessive recruitment of FUS and heterogeneous nuclear ribonucleoprotein K (HNRNPK). Our findings that dendritic spine loss is because of excess recruitment of RNA-binding proteins provide a basis for the future exploration of ALS-related RNA-binding proteins.
Assuntos
Esclerose Lateral Amiotrófica , Sarcoma , Masculino , Feminino , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Regiões 3' não Traduzidas/genética , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Espinhas Dendríticas/metabolismo , Mutação , Proteínas de Ligação a RNA/genética , RNA Mensageiro/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Sarcoma/genética , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
Translational control of mRNAs in dendrites is essential for certain forms of synaptic plasticity and learning and memory. CPEB is an RNA-binding protein that regulates local translation in dendrites. Here, we identify poly(A) polymerase Gld2, deadenylase PARN, and translation inhibitory factor neuroguidin (Ngd) as components of a dendritic CPEB-associated polyadenylation apparatus. Synaptic stimulation induces phosphorylation of CPEB, PARN expulsion from the ribonucleoprotein complex, and polyadenylation in dendrites. A screen for mRNAs whose polyadenylation is altered by Gld2 depletion identified >100 transcripts including one encoding NR2A, an NMDA receptor subunit. shRNA depletion studies demonstrate that Gld2 promotes and Ngd inhibits dendritic NR2A expression. Finally, shRNA-mediated depletion of Gld2 in vivo attenuates protein synthesis-dependent long-term potentiation (LTP) at hippocampal dentate gyrus synapses; conversely, Ngd depletion enhances LTP. These results identify a pivotal role for polyadenylation and the opposing effects of Gld2 and Ngd in hippocampal synaptic plasticity.
Assuntos
Citoplasma/metabolismo , Plasticidade Neuronal , Biossíntese de Proteínas , Transmissão Sináptica , Animais , Dendritos/metabolismo , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Poliadenilação , Polinucleotídeo Adenililtransferase/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Repressoras/metabolismo , Ribonucleoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
During early embryonic development, the RNA-binding protein CPEB mediates cytoplasmic polyadenylation and translational activation through a combinatorial code defined by the cy-toplasmic polyadenylation element (CPE) present in maternal mRNAs. However, in non-neuronal somatic cells, CPEB accelerates deadenylation to repress translation of the target, including c-myc mRNA, through an ill-defined cis-regulatory mechanism. Using RNA mutagenesis and electrophoretic mobility shift assays, we demonstrated that a combination of tandemly arranged consensus (cCPE) and non-consensus (ncCPE) cytoplasmic polyadenylation elements (CPEs) constituted a combinatorial code for CPEB-mediated c-myc mRNA decay. CPEB binds to cCPEs with high affinity (Kd = ~250 nM), whereas it binds to ncCPEs with low affinity (Kd > ~900 nM). CPEB binding to a cCPE enhances CPEB binding to the proximal ncCPE. In contrast, while a cCPE did not activate mRNA degradation, an ncCPE was essential for the induction of degradation, and a combination of a cCPE and ncCPEs further promoted degradation. Based on these findings, we propose a model in which the high-affinity binding of CPEB to the cCPE accelerates the binding of the second CPEB to the ncCPEs, resulting in the recruitment of deadenylases, acceleration of deadenylation, and repression of c-myc mRNAs.
Assuntos
Oócitos , Fatores de Poliadenilação e Clivagem de mRNA , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Oócitos/metabolismo , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , HumanosRESUMO
In eukaryotes, the 40 S ribosomal subunit serves as the platform of initiation factor assembly, to place itself precisely on the AUG start codon. Structural arrangement of the 18 S rRNA determines the overall shape of the 40 S subunit. Here, we present genetic evaluation of yeast 18 S rRNA function using 10 point mutations altering the polysome profile. All the mutants reduce the abundance of the mutant 40 S, making it limiting for translation initiation. Two of the isolated mutations, G875A, altering the core of the platform domain that binds eIF1 and eIF2, and A1193U, changing the h31 loop located below the P-site tRNA(i)(Met), show phenotypes indicating defective regulation of AUG selection. Evidence is provided that these mutations reduce the interaction with the components of the preinitiation complex, thereby inhibiting its function at different steps. These results indicate that the 18 S rRNA mutations impair the integrity of scanning-competent preinitiation complex, thereby altering the 40 S subunit response to stringent AUG selection. Interestingly, nine of the mutations alter the body/platform domains of 18 S rRNA, potentially affecting the bridges to the 60 S subunit, but they do not change the level of 18 S rRNA intermediates. Based on these results, we also discuss the mechanism of the selective degradation of the mutant 40 S subunits.
Assuntos
Códon de Iniciação/metabolismo , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Subunidades Proteicas/metabolismo , RNA Fúngico , RNA Ribossômico 18S , Subunidades Ribossômicas Menores de Eucariotos , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , Mutação Puntual , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Here, we describe a protocol for tRNA identification in the 60S ribosome-nascent peptide complex co-purified with Nuclear Export Mediator Factor (NEMF), a responsible factor for C-terminal alanine and threonine tailing of the nascent peptide. Our protocol is based on regular reverse transcription followed by quantitative Polymerase chain reaction (PCR). Although this method cannot distinguish between amino acid-charged and uncharged and base-modified and unmodified tRNAs, it is a convenient way to estimate the relative level of tRNA species and thus can be useful for researchers. For complete details on the use and execution of this protocol, please refer to Udagawa et al. (2021).
Assuntos
RNA de Transferência/metabolismo , Ribossomos/metabolismo , Células HEK293 , Humanos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Ribosome-associated quality control (RQC) relieves stalled ribosomes and eliminates potentially toxic nascent polypeptide chains (NCs) that can cause neurodegeneration. During RQC, RQC2 modifies NCs with a C-terminal alanine and threonine (CAT) tail. CAT tailing promotes ubiquitination of NCs for proteasomal degradation, while RQC failure in budding yeast disrupts proteostasis via CAT-tailed NC aggregation. However, the CAT tail and its cytotoxicity in mammals have remained largely uncharacterized. We demonstrate that NEMF, a mammalian RQC2 homolog, modifies translation products of nonstop mRNAs, major erroneous mRNAs in mammals, with a C-terminal tail mainly composed of alanine with several other amino acids. Overproduction of nonstop mRNAs induces NC aggregation and caspase-3-dependent apoptosis and impairs neuronal morphogenesis, which are ameliorated by NEMF depletion. Moreover, we found that homopolymeric alanine tailing at least partially accounts for CAT-tail cytotoxicity. These findings explain the cytotoxicity of CAT-tailed NCs and demonstrate physiological significance of RQC on proper neuronal morphogenesis and cell survival.
Assuntos
Antígenos de Neoplasias/metabolismo , Neurônios/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Alanina/metabolismo , Linhagem Celular , Sobrevivência Celular , Células HEK293 , Células HeLa , Humanos , Morfogênese , Peptídeos/metabolismo , Biossíntese de Proteínas , Proteólise , Treonina/metabolismo , UbiquitinaçãoRESUMO
Ribosome movement is not always smooth and is rather often impeded. For ribosome pauses, fundamental issues remain to be addressed, including where ribosomes pause on mRNAs, what kind of RNA/amino acid sequence causes this pause, and the physiological significance of this attenuation of protein synthesis. Here, we survey the positions of ribosome collisions caused by ribosome pauses in humans and zebrafish using modified ribosome profiling. Collided ribosomes, i.e., disomes, emerge at various sites: Pro-Pro/Gly/Asp motifs; Arg-X-Lys motifs; stop codons; and 3' untranslated regions. The electrostatic interaction between the charged nascent chain and the ribosome exit tunnel determines the eIF5A-mediated disome rescue at the Pro-Pro sites. In particular, XBP1u, a precursor of endoplasmic reticulum (ER)-stress-responsive transcription factor, shows striking queues of collided ribosomes and thus acts as a degradation substrate by ribosome-associated quality control. Our results provide insight into the causes and consequences of ribosome pause by dissecting collided ribosomes.
Assuntos
Códon de Terminação/genética , Biossíntese de Proteínas/genética , Ribossomos/genética , Ribossomos/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Códon de Terminação/metabolismo , Humanos , Elongação Traducional da Cadeia Peptídica/genética , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Peixe-ZebraRESUMO
eIF2α phosphorylation-mediated translational regulation is crucial for global translation repression by various stresses, including the unfolded protein response (UPR). However, translational control during UPR has not been demonstrated in yeast. This study investigated ribosome ubiquitination-mediated translational controls during UPR. Tunicamycin-induced ER stress enhanced the levels of ubiquitination of the ribosomal proteins uS10, uS3 and eS7. Not4-mediated monoubiquitination of eS7A was required for resistance to tunicamycin, whereas E3 ligase Hel2-mediated ubiquitination of uS10 was not. Ribosome profiling showed that the monoubiquitination of eS7A was crucial for translational regulation, including the upregulation of the spliced form of HAC1 (HAC1i) mRNA and the downregulation of Histidine triad NucleoTide-binding 1 (HNT1) mRNA. Downregulation of the deubiquitinating enzyme complex Upb3-Bre5 increased the levels of ubiquitinated eS7A during UPR in an Ire1-independent manner. These findings suggest that the monoubiquitination of ribosomal protein eS7A plays a crucial role in translational controls during the ER stress response in yeast.
Assuntos
Estresse do Retículo Endoplasmático , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Endopeptidases/metabolismo , Regulação Fúngica da Expressão Gênica , Hidrolases/genética , Hidrolases/metabolismo , Biossíntese de Proteínas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Resposta a Proteínas não DobradasRESUMO
In eukaryotic translation initiation, eIF2GTP-Met-tRNA(i)(Met) ternary complex (TC) interacts with eIF3-eIF1-eIF5 complex to form the multifactor complex (MFC), while eIF2GDP associates with eIF2B for guanine nucleotide exchange. Gcn2p phosphorylates eIF2 to inhibit eIF2B. Here we evaluate the abundance of eIFs and their pre-initiation intermediate complexes in gcn2 deletion mutant grown under different conditions. We show that ribosomes are three times as abundant as eIF1, eIF2 and eIF5, while eIF3 is half as abundant as the latter three and hence, the limiting component in MFC formation. By quantitative immunoprecipitation, we estimate that approximately 15% of the cellular eIF2 is found in TC during rapid growth in a complex rich medium. Most of the TC is found in MFC, and important, approximately 40% of the total eIF2 is associated with eIF5 but lacks tRNA(i)(Met). When the gcn2Delta mutant grows less rapidly in a defined complete medium, TC abundance increases threefold without altering the abundance of each individual factor. Interestingly, the TC increase is suppressed by eIF5 overexpression and Gcn2p expression. Thus, eIF2B-catalyzed TC formation appears to be fine-tuned by eIF2 phosphorylation and the novel eIF2/eIF5 complex lacking tRNA(i)(Met).
Assuntos
Iniciação Traducional da Cadeia Peptídica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Meios de Cultura , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 5 em Eucariotos/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Biossíntese de Proteínas , RNA de Transferência de Metionina/metabolismo , Ribossomos/metabolismoRESUMO
This chapter describes phenotypic assays on specific and general aspects of translation using yeast Saccharomyces cerevisiae as a model eukaryote. To study the effect on start codon selection stringency, a his4(-) or his4-lacZ allele altering the first AUG to AUU is employed. Mutations relaxing the stringent selection confer the His(+) phenotype in the his4(-) strain background or increase expression from his4-lacZ compared to that from wild-type HIS4-lacZ (Sui(-) phenotype). Translation of the Gcn4p transcription activator is strictly regulated by amino acid availability depending on upstream ORF (uORF) elements in the GCN4 mRNA leader. Mutations reducing the eIF2/GTP/Met-tRNA(i)(Met) complex level or the rate of its binding to the 40S subunit derepress GCN4 translation by allowing ribosomes to bypass inhibitory uORFs in the absence of the starvation signal (Gcd(-) phenotype). Mutations impairing scanning or AUG recognition generally impair translational GCN4 induction during amino acid starvation (Gcn(-) phenotype). Different amino acid analogs or amino acid enzyme inhibitors are used to study Gcd(-) or Gcn(-) phenotypes. The method of polysome profiling is also described to gain an ultimate "phenotypic" proof for translation defects.
Assuntos
Iniciação Traducional da Cadeia Peptídica/fisiologia , Biossíntese de Proteínas/fisiologia , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica , Centrifugação com Gradiente de Concentração , Códon de Iniciação , Proteínas de Ligação a DNA/fisiologia , Vetores Genéticos , Fenótipo , Polirribossomos/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/fisiologia , Transformação GenéticaRESUMO
FUS is an RNA-binding protein associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Previous reports have demonstrated intrinsic roles of FUS in synaptic function. However, the mechanism underlying FUS's regulation of synaptic morphology has remained unclear. We found that reduced mature spines after FUS depletion were associated with the internalization of PSD-95 within the dendritic shaft. Mass spectrometry of PSD-95-interacting proteins identified SynGAP, whose expression decreased after FUS depletion. Moreover, FUS and the ELAV-like proteins ELAVL4 and ELAVL1 control SynGAP mRNA stability in a 3'UTR length-dependent manner, resulting in the stable expression of the alternatively spliced SynGAP isoform α2. Finally, abnormal spine maturation and FTLD-like behavioral deficits in FUS-knockout mice were ameliorated by SynGAP α2. Our findings establish an important link between FUS and ELAVL proteins for mRNA stability control and indicate that this mechanism is crucial for the maintenance of synaptic morphology and cognitive function.
Assuntos
Regiões 3' não Traduzidas , Cognição/fisiologia , Espinhas Dendríticas/fisiologia , Proteínas ELAV/genética , RNA Mensageiro/metabolismo , Proteína FUS de Ligação a RNA/genética , Proteínas Ativadoras de ras GTPase/genética , Animais , Espinhas Dendríticas/metabolismo , Proteínas ELAV/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Isoformas de Proteínas , RNA Mensageiro/genética , Proteína FUS de Ligação a RNA/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
Translation arrest by polybasic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system. Here we report that ubiquitination of the 40S ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 (or RQT1) is required for RQC. We identify a RQC-trigger (RQT) subcomplex composed of the RNA helicase-family protein Slh1/Rqt2, the ubiquitin-binding protein Cue3/Rqt3, and yKR023W/Rqt4 that is required for RQC. The defects in RQC of the RQT mutants correlate with sensitivity to anisomycin, which stalls ribosome at the rotated form. Cryo-electron microscopy analysis reveals that Hel2-bound ribosome are dominantly the rotated form with hybrid tRNAs. Ribosome profiling reveals that ribosomes stalled at the rotated state with specific pairs of codons at P-A sites serve as RQC substrates. Rqt1 specifically ubiquitinates these arrested ribosomes to target them to the RQT complex, allowing subsequent RQC reactions including dissociation of the stalled ribosome into subunits.Several protein quality control mechanisms are in place to trigger the rapid degradation of aberrant polypeptides and mRNAs. Here the authors describe a mechanism of ribosome-mediated quality control that involves the ubiquitination of ribosomal proteins by the E3 ubiquitin ligase Hel2/RQT1.
Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Ribossomos/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Células HEK293 , Humanos , Mutação , Biossíntese de Proteínas , Conformação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , UbiquitinaçãoRESUMO
Apoptosis signal-regulating kinase 1 (ASK1) is an oxidative stress-responsive kinase that is regulated by various interacting molecules and post-translational modifications. However, how these molecules and modifications cooperatively regulate ASK1 activity remains largely unknown. Here, we showed that tripartite motif 48 (TRIM48) orchestrates the regulation of oxidative stress-induced ASK1 activation. A pull-down screen identified a TRIM48-interacting partner, protein arginine methyltransferase 1 (PRMT1), which negatively regulates ASK1 activation by enhancing its interaction with thioredoxin (Trx), another ASK1-negative regulator. TRIM48 facilitates ASK1 activation by promoting K48-linked polyubiquitination and degradation of PRMT1. TRIM48 knockdown suppressed oxidative stress-induced ASK1 activation and cell death, whereas forced expression promoted cancer cell death in mouse xenograft model. These results indicate that TRIM48 facilitates oxidative stress-induced ASK1 activation and cell death through ubiquitination-dependent degradation of PRMT1. This study provides a cell death mechanism fine-tuned by the crosstalk between enzymes that engage various types of post-translational modifications.
Assuntos
Morte Celular/fisiologia , MAP Quinase Quinase Quinase 5/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Apoptose/genética , Apoptose/fisiologia , Morte Celular/genética , Linhagem Celular , Humanos , MAP Quinase Quinase Quinase 5/genética , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/genética , Proteínas com Motivo Tripartido/genética , Ubiquitina/metabolismo , Ubiquitinação/genética , Ubiquitinação/fisiologiaRESUMO
Fused in sarcoma (FUS) and splicing factor, proline- and glutamine-rich (SFPQ) are RNA binding proteins that regulate RNA metabolism. We found that alternative splicing of the Mapt gene at exon 10, which generates 4-repeat tau (4R-T) and 3-repeat tau (3R-T), is regulated by interactions between FUS and SFPQ in the nuclei of neurons. Hippocampus-specific FUS- or SFPQ-knockdown mice exhibit frontotemporal lobar degeneration (FTLD)-like behaviors, reduced adult neurogenesis, accumulation of phosphorylated tau, and hippocampal atrophy with neuronal loss through an increased 4R-T/3R-T ratio. Normalization of this increased ratio by 4R-T-specific silencing results in recovery of the normal phenotype. These findings suggest a biological link among FUS/SFPQ, tau isoform alteration, and phenotypic expression, which may function in the early pathomechanism of FTLD.
Assuntos
Degeneração Lobar Frontotemporal/metabolismo , Fator de Processamento Associado a PTB/metabolismo , Isoformas de Proteínas/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Proteínas tau/metabolismo , Processamento Alternativo/fisiologia , Animais , Éxons/fisiologia , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Fenótipo , Splicing de RNA/fisiologia , Proteínas de Ligação a RNA/metabolismoRESUMO
FUS is an RNA/DNA-binding protein involved in multiple steps of gene expression and is associated with amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD). However, the specific disease-causing and/or modifying mechanism mediated by FUS is largely unknown. Here we evaluate intrinsic roles of FUS on synaptic functions and animal behaviours. We find that FUS depletion downregulates GluA1, a subunit of AMPA receptor. FUS binds GluA1 mRNA in the vicinity of the 3' terminus and controls poly (A) tail maintenance, thus regulating stability. GluA1 reduction upon FUS knockdown reduces miniature EPSC amplitude both in cultured neurons and in vivo. FUS knockdown in hippocampus attenuates dendritic spine maturation and causes behavioural aberrations including hyperactivity, disinhibition and social interaction defects, which are partly ameliorated by GluA1 reintroduction. These results highlight the pivotal role of FUS in regulating GluA1 mRNA stability, post-synaptic function and FTLD-like animal behaviours.
Assuntos
Neurônios/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Receptores de AMPA/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Comportamento Animal/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/metabolismo , Regulação da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Hipocampo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína FUS de Ligação a RNA/genética , Receptores de AMPA/genéticaRESUMO
[This corrects the article DOI: 10.1016/j.fob.2013.11.001.].
RESUMO
IMPORTANCE: TAR DNA-binding protein of 43 kDa (TDP-43) plays a major role in the pathogenesis of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Although a pathological continuity between FTLD and ALS has been suggested, the neuropathological changes of the lower motor neuron (LMN) systems have not been assessed in TDP-43-associated FTLD (FTLD-TDP), to our knowledge. OBJECTIVE: To investigate a pathological continuity between FTLD-TDP and ALS by comparing their respective neuropathological changes in the motor neuron system. DESIGN AND SETTING: A retrospective clinical medical record review and a semiquantitative neuropathological evaluation of the cranial motor nerve nuclei and spinal cord were conducted at autopsy. We included 43 patients with sporadic FTLD-TDP, type A, B, or C, from 269 consecutively autopsied patients with TDP-43 proteinopathy. Patients were categorized as having FTLD without ALS, FTLD-ALS (onset of FTLD symptoms/signs preceded those of ALS), or ALS-FTLD (onset of ALS symptoms/signs preceded those of FTLD). MAIN OUTCOMES AND MEASURES: Neuronal TDP-43 pathological changes and neuronal loss. RESULTS: Forty-three patients were included in the clinical analysis, and 29 from whom spinal cords were obtained were included in the neuropathological analysis. Survival time was significantly shorter in the FTLD-ALS and ALS-FTLD groups than in the FTLD without ALS group (P < .001). At neuropathological examination, 89% of patients in the FTLD without ALS group showed aggregations of TDP-43 in the spinal motor neurons. The LMN loss was most severe in ALS-FTLD, followed by FTLD-ALS and FTLD without ALS. All the patients with type A or C FTLD-TDP were included in the FTLD without ALS group, and all those with type B pathological changes were in the FTLD-ALS or the ALS-FTLD group. Lower motor neuron loss and TDP-43-positive skeinlike inclusions were observed in all pathological subtypes. CONCLUSIONS AND RELEVANCE: The LMN systems of FTLD-TDP frequently exhibit neuropathological changes corresponding to ALS. Thus, a pathological continuity between FTLD-TDP and ALS is supported at the level of the LMN system.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Proteínas de Ligação a DNA/análise , Degeneração Lobar Frontotemporal/patologia , Doença dos Neurônios Motores/patologia , Idoso , Esclerose Lateral Amiotrófica/genética , Proteínas de Ligação a DNA/genética , Feminino , Degeneração Lobar Frontotemporal/genética , Humanos , Corpos de Inclusão/química , Corpos de Inclusão/patologia , Masculino , Pessoa de Meia-Idade , Doença dos Neurônios Motores/genética , Estudos RetrospectivosRESUMO
FUS is genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). To clarify the RNA metabolism cascade regulated by FUS in ALS/FTLD, we compared the FUS-regulated transcriptome profiles in different lineages of primary cells from the central nervous system. The profiles of FUS-mediated gene expression and alternative splicing in motor neurons were similar to those of cortical neurons, but not to those in cerebellar neurons despite the similarity of innate transcriptome signature. The gene expression profiles in glial cells were similar to those in motor and cortical neurons. We identified certain neurological diseases-associated genes, including Mapt, Stx1a, and Scn8a, among the profiles of gene expression and alternative splicing events regulated by FUS. Thus, FUS-regulated transcriptome profiles in each cell-type may determine cellular fate in association with FUS-mediated ALS/FTLD, and identified RNA targets for FUS could be therapeutic targets for ALS/FTLD.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Encéfalo/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Transcriptoma , Regulação da Expressão Gênica , Humanos , Neurônios/patologia , Distribuição TecidualRESUMO
In yeast, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During the last step of translation initiation, eukaryotic initiation factor 5B (eIF5B) promotes the 60S subunit joining with the 40S initiation complex (IC). Malfunctional 60S subunits produced by misfolding or mutation may disrupt the 40S IC stalling on the start codon, thereby altering the stringency of initiation. Using several point mutations isolated by random mutagenesis, here we studied the role of 25S rRNA in start codon selection. Three mutations changing bases near the ribosome surface had strong effects, allowing the initiating ribosomes to skip both AUG and non-AUG codons: C2879U and U2408C, altering the A loop and P loop, respectively, of the peptidyl transferase center, and G1735A, mapping near a Eukarya-specific bridge to the 40S subunit. Overexpression of eIF5B specifically suppressed the phenotype caused by C2879U, suggesting functional interaction between eIF5B and the A loop. In vitro reconstitution assays showed that C2879U decreased eIF5B-catalyzed 60S subunit joining with a 40S IC. Thus, eIF5B interaction with the peptidyl transferase center A loop increases the accuracy of initiation by stabilizing the overall conformation of the 80S initiation complex. This study provides an insight into the effect of ribosomal mutations on translation profiles in eukaryotes.
Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , RNA Fúngico/química , RNA Fúngico/metabolismo , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Códon de Iniciação/genética , Códon de Iniciação/metabolismo , Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/genética , Genes Fúngicos , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Iniciação Traducional da Cadeia Peptídica , Peptidil Transferases/química , Peptidil Transferases/metabolismo , Conformação Proteica , RNA Fúngico/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico/genética , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
TDP-43 and FUS are linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), and loss of function of either protein contributes to these neurodegenerative conditions. To elucidate the TDP-43- and FUS-regulated pathophysiological RNA metabolism cascades, we assessed the differential gene expression and alternative splicing profiles related to regulation by either TDP-43 or FUS in primary cortical neurons. These profiles overlapped by >25% with respect to gene expression and >9% with respect to alternative splicing. The shared downstream RNA targets of TDP-43 and FUS may form a common pathway in the neurodegenerative processes of ALS/FTLD.