RESUMEN
About 150 post-transcriptional RNA modifications have been identified in all kingdoms of life. During RNA catabolism, most modified nucleosides are resistant to degradation and are released into the extracellular space. In this study, we explored the physiological role of these extracellular modified nucleosides and found that N6-methyladenosine (m6A), widely recognized as an epigenetic mark in RNA, acts as a ligand for the human adenosine A3 receptor, for which it has greater affinity than unmodified adenosine. We used structural modeling to define the amino acids required for specific binding of m6A to the human A3 receptor. We also demonstrated that m6A was dynamically released in response to cytotoxic stimuli and facilitated type I allergy in vivo. Our findings implicate m6A as a signaling molecule capable of activating G protein-coupled receptors (GPCRs) and triggering pathophysiological responses, a previously unreported property of RNA modifications.
Asunto(s)
Adenosina/análogos & derivados , Epigénesis Genética , Procesamiento Postranscripcional del ARN , Receptor de Adenosina A3/metabolismo , Transducción de Señal , Adenosina/genética , Adenosina/metabolismo , Animales , Femenino , Células HEK293 , Humanos , Masculino , Conejos , Receptor de Adenosina A3/genéticaRESUMEN
Naked mole-rats (NMRs) have exceptional longevity and are resistant to age-related physiological decline and diseases. Given the role of cellular senescence in aging, we postulated that NMRs possess unidentified species-specific mechanisms to prevent senescent cell accumulation. Here, we show that upon induction of cellular senescence, NMR fibroblasts underwent delayed and progressive cell death that required activation of the INK4a-retinoblastoma protein (RB) pathway (termed "INK4a-RB cell death"), a phenomenon not observed in mouse fibroblasts. Naked mole-rat fibroblasts uniquely accumulated serotonin and were inherently vulnerable to hydrogen peroxide (H2 O2 ). After activation of the INK4a-RB pathway, NMR fibroblasts increased monoamine oxidase levels, leading to serotonin oxidization and H2 O2 production, which resulted in increased intracellular oxidative damage and cell death activation. In the NMR lung, induction of cellular senescence caused delayed, progressive cell death mediated by monoamine oxidase activation, thereby preventing senescent cell accumulation, consistent with in vitro results. The present findings indicate that INK4a-RB cell death likely functions as a natural senolytic mechanism in NMRs, providing an evolutionary rationale for senescent cell removal as a strategy to resist aging.
Asunto(s)
Senescencia Celular , Serotonina , Animales , Ratones , Serotonina/metabolismo , Senescencia Celular/fisiología , Envejecimiento/metabolismo , Muerte Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Ratas Topo/metabolismoRESUMEN
MTU1 controls intramitochondrial protein synthesis by catalyzing the 2-thiouridine modification of mitochondrial transfer RNAs (mt-tRNAs). Missense mutations in the MTU1 gene are associated with life-threatening reversible infantile hepatic failure. However, the molecular pathogenesis is not well understood. Here, we investigated 17 mutations associated with this disease, and our results showed that most disease-related mutations are partial loss-of-function mutations, with three mutations being particularly severe. Mutant MTU1 is rapidly degraded by mitochondrial caseinolytic peptidase (CLPP) through a direct interaction with its chaperone protein CLPX. Notably, knockdown of CLPP significantly increased mutant MTU1 protein expression and mt-tRNA 2-thiolation, suggesting that accelerated proteolysis of mutant MTU1 plays a role in disease pathogenesis. In addition, molecular dynamics simulations demonstrated that disease-associated mutations may lead to abnormal intermolecular interactions, thereby impairing MTU1 enzyme activity. Finally, clinical data analysis underscores a significant correlation between patient prognosis and residual 2-thiolation levels, which is partially consistent with the AlphaMissense predictions. These findings provide a comprehensive understanding of MTU1-related diseases, offering prospects for modification-based diagnostics and novel therapeutic strategies centered on targeting CLPP.
Asunto(s)
Mitocondrias , Proteínas Mitocondriales , Péptido Hidrolasas , ARNt Metiltransferasas , Humanos , Endopeptidasa Clp/genética , Endopeptidasa Clp/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Mutación , Péptido Hidrolasas/genética , Proteolisis , ARN Mitocondrial/metabolismo , ARN de Transferencia/metabolismo , ARNt Metiltransferasas/genética , Proteínas Mitocondriales/metabolismoRESUMEN
In higher eukaryotes, tRNA methyltransferase 10A (TRMT10A) is responsible for N1-methylguanosine modification at position nine of various cytoplasmic tRNAs. Pathogenic mutations in TRMT10A cause intellectual disability, microcephaly, diabetes, and short stature in humans, and generate cytotoxic tRNA fragments in cultured cells; however, it is not clear how TRMT10A supports codon translation or brain functions. Here, we generated Trmt10a null mice and showed that tRNAGln(CUG) and initiator methionine tRNA levels were universally decreased in various tissues; the same was true in a human cell line lacking TRMT10A. Ribosome profiling of mouse brain revealed that dysfunction of TRMT10A causes ribosome slowdown at the Gln(CAG) codon and increases translation of Atf4 due to higher frequency of leaky scanning of its upstream open reading frames. Broadly speaking, translation of a subset of mRNAs, especially those for neuronal structures, is perturbed in the mutant brain. Despite not showing discernable defects in the pancreas, liver, or kidney, Trmt10a null mice showed lower body weight and smaller hippocampal postsynaptic densities, which is associated with defective synaptic plasticity and memory. Taken together, our study provides mechanistic insight into the roles of TRMT10A in the brain, and exemplifies the importance of universal tRNA modification during translation of specific codons.
RESUMEN
N6 -isopentenyladenosine (i6A), a modified adenosine monomer, is known to induce cell death upon its addition to the culture medium. However, the molecular fate of extracellularly added i6A has yet to be identified. Here we show that i6A addition to cell culture medium results in i6A incorporation into cellular RNA in several cell lines, including the 5-fluorouracil (5-FU)-resistant human oral squamous cell carcinoma cell line FR2-SAS and its parental 5-FU-sensitive cell line SAS. i6A was predominantly incorporated into 18S and 28S rRNAs, and i6A incorporation into total RNA was mostly suppressed by treating these cell lines with an RNA polymerase I (Pol I) inhibitor. i6A was incorporated into RNA even upon inactivation of TRIT1, the only cellular i6A-modifying enzyme. These results indicate that upon cellular uptake of i6A, it is anabolized to be used for Pol I transcription. Interestingly, at lower i6A concentrations, the cytotoxic effect of i6A was substantially more pronounced in FR2-SAS cells than in SAS cells. Moreover, in FR2-SAS cells, i6A treatment decreased the rate of cellular protein synthesis and increased intracellular protein aggregation, and these effects were more pronounced than in SAS cells. Our work provides insights into the molecular fate of extracellularly applied i6A in the context of intracellular nucleic acid anabolism and suggests investigation of i6A as a candidate for a chemotherapy agent against 5-FU-resistant cancer cells.
Asunto(s)
Antineoplásicos , Carcinoma de Células Escamosas , Neoplasias de la Boca , Línea Celular Tumoral , Fluorouracilo/metabolismo , Fluorouracilo/farmacología , Humanos , Isopenteniladenosina , ARN , ARN Ribosómico/metabolismoRESUMEN
Understanding the physiology of human-induced pluripotent stem cells (iPSCs) is necessary for directed differentiation, mimicking embryonic development, and regenerative medicine applications. Pluripotent stem cells (PSCs) exhibit unique abilities such as self-renewal and pluripotency, but they lack some functions that are associated with normal somatic cells. One such function is the circadian oscillation of clock genes; however, whether or not PSCs demonstrate this capability remains unclear. In this study, the reason why circadian rhythm does not oscillate in human iPSCs was examined. This phenomenon may be due to the transcriptional repression of clock genes resulting from the hypermethylation of histone H3 at lysine 27 (H3K27), or it may be due to the low levels of brain and muscle ARNT-like 1 (BMAL1) protein. Therefore, BMAL1-overexpressing cells were generated and pre-treated with GSK126, an inhibitor of enhancer of zest homologue 2 (EZH2), which is a methyltransferase of H3K27 and a component of polycomb repressive complex 2. Consequently, a significant circadian rhythm following endogenous BMAL1, period 2 (PER2), and other clock gene expression was induced by these two factors, suggesting a candidate mechanism for the lack of rhythmicity of clock gene expression in iPSCs.
Asunto(s)
Factores de Transcripción ARNTL , Relojes Circadianos , Células Madre Pluripotentes Inducidas , Complejo Represivo Polycomb 2 , Humanos , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Encéfalo/metabolismo , Relojes Circadianos/genética , Ritmo Circadiano/fisiología , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Músculos/metabolismo , Complejo Represivo Polycomb 2/metabolismoRESUMEN
Human endogenous retroviruses (HERVs) occupy approximately 8% of the human genome. HERVs, transcribed in early embryos, are epigenetically silenced in somatic cells, except under pathological conditions. HERV-K is thought to protect embryos from exogenous viral infection. However, uncontrolled HERV-K expression in somatic cells has been implicated in several diseases. Here, we show that SOX2, which plays a key role in maintaining the pluripotency of stem cells, is critical for HERV-K LTR5Hs. HERV-K undergoes retrotransposition within producer cells in the absence of Env expression. Furthermore, we identified new HERV-K integration sites in long-term culture of induced pluripotent stem cells that express SOX2. These results suggest that the strict dependence of HERV-K on SOX2 has allowed HERV-K to protect early embryos during evolution while limiting the potentially harmful effects of HERV-K retrotransposition on host genome integrity in these early embryos. IMPORTANCE Human endogenous retroviruses (HERVs) account for approximately 8% of the human genome; however, the physiological role of HERV-K remains unknown. This study found that HERV-K LTR5Hs and LTR5B were transactivated by SOX2, which is essential for maintaining and reestablishing pluripotency. HERV-K can undergo retrotransposition within producer cells without env expression, and new integration sites may affect cell proliferation. In induced pluripotent stem cells (iPSCs), genomic impairment due to HERV-K retrotransposition has been identified, but it is a rare event. Considering the retention of SOX2-responsive elements in the HERV-K long terminal repeat (LTR) for over 20 million years, we conclude that HERV-K may play important physiological roles in SOX2-expressing cells.
Asunto(s)
Retrovirus Endógenos , Células Madre Pluripotentes Inducidas , Factores de Transcripción SOXB1 , Retrovirus Endógenos/genética , Humanos , Células Madre Pluripotentes Inducidas/virología , Factores de Transcripción SOXB1/genética , Secuencias Repetidas Terminales/genética , Integración ViralRESUMEN
Retroviral infection requires reverse transcription, and the reverse transcriptase (RT) uses cellular tRNA as its primer. In humans, the TRMT6-TRMT61A methyltransferase complex incorporates N1-methyladenosine modification at tRNA position 58 (m1A58); however, the role of m1A58 as an RT-stop site during retroviral infection has remained questionable. Here, we constructed TRMT6 mutant cells to determine the roles of m1A in HIV-1 infection. We confirmed that tRNA3Lys m1A58 was required for in vitro plus-strand strong-stop by RT. Accordingly, infectivity of VSV-G pseudotyped HIV-1 decreased when the virus contained m1A58-deficient tRNA3Lys instead of m1A58-modified tRNA3Lys. In TRMT6 mutant cells, the global protein synthesis rate was equivalent to that of wild-type cells. However, unexpectedly, plasmid-derived HIV-1 expression showed that TRMT6 mutant cells decreased accumulation of HIV-1 capsid, integrase, Tat, Gag, and GagPol proteins without reduction of HIV-1 RNAs in cells, and fewer viruses were produced. Moreover, the importance of 5,2'-O-dimethyluridine at U54 of tRNA3Lys as a second RT-stop site was supported by conservation of retroviral genome-tRNALys sequence-complementarity, and TRMT6 was required for efficient 5-methylation of U54. These findings illuminate the fundamental importance of tRNA m1A58 modification in both the early and late steps of HIV-1 replication, as well as in the cellular tRNA modification network.
Asunto(s)
VIH-1/fisiología , Procesamiento Postranscripcional del ARN , ARN de Transferencia de Lisina/metabolismo , Replicación Viral , Animales , Células HEK293 , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Metilación , Ratones , Mutación , ARN de Transferencia de Lisina/químicaRESUMEN
RNA contains a wide variety of posttranscriptional modifications covalently attached to its base or sugar group. These modified nucleosides are liberated from RNA molecules as the consequence of RNA catabolism and released into extracellular space, but the molecular mechanism of extracellular transport and its pathophysiological implications have been unclear. In the present study, we discovered that RNA-derived modified nucleosides are exported to extracellular space through equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2), with ENT1 showing higher preference for modified nucleosides than ENT2. Pharmacological inhibition or genetic deletion of ENT1 and ENT2 significantly attenuated export of modified nucleosides thereby resulting in their accumulation in cytosol. Using mutagenesis strategy, we identified an amino acid residue in ENT1 that is involved in the discrimination of unmodified and modified nucleosides. In ENTs-deficient cells, the elevated levels of intracellular modified nucleosides were closely associated with an induction of autophagy response as evidenced by increased LC3-II level. Importantly, we performed a screening of modified nucleosides capable of inducing autophagy and found that 1-methylguanosine (m1G) was sufficient to induce LC3-II levels. Pathophysiologically, defective export of modified nucleosides drastically induced Zika virus replication in an autophagy-dependent manner. In addition, we also found that pharmacological inhibition of ENTs by dilazep significantly induced Zika virus replication. Collectively, our findings highlight RNA-derived modified nucleosides as important signaling modulators that activate autophagy response and indicate that defective export of these modified nucleoside can have profound consequences for pathophysiology.
Asunto(s)
Autofagia , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Transportador Equilibrativo 2 de Nucleósido/metabolismo , Nucleósidos/metabolismo , ARN/metabolismo , Infección por el Virus Zika/virología , Virus Zika/fisiología , Transporte Activo de Núcleo Celular , Tranportador Equilibrativo 1 de Nucleósido/genética , Transportador Equilibrativo 2 de Nucleósido/genética , Humanos , Nucleósidos/química , Nucleósidos/genética , ARN/genética , Células Tumorales Cultivadas , Replicación Viral , Infección por el Virus Zika/genética , Infección por el Virus Zika/patologíaRESUMEN
Post-transcriptional modifications in mitochondrial tRNAs (mt-tRNAs) play critical roles in mitochondrial protein synthesis, which produces respiratory chain complexes. In this study, we took advantage of mass spectrometric analysis to map 5-methylcytidine (m5C) at positions 48-50 in eight mouse and six human mt-tRNAs. We also confirmed the absence of m5C in mt-tRNAs isolated from Nsun2 knockout (KO) mice, as well as from NSUN2 KO human culture cells. In addition, we successfully reconstituted m5C at positions 48-50 of mt-tRNA in vitro with NSUN2 protein in the presence of S-adenosylmethionine. Although NSUN2 is predominantly localized to the nucleus and introduces m5C into cytoplasmic tRNAs and mRNAs, structured illumination microscopy clearly revealed NSUN2 foci inside mitochondria. These observations provide novel insights into the role of NSUN2 in the physiology and pathology of mitochondrial functions.
Asunto(s)
5-Metilcitosina/metabolismo , Metiltransferasas/genética , Mitocondrias/genética , Procesamiento Postranscripcional del ARN , ARN Mitocondrial/genética , ARN de Transferencia/genética , Animales , Sistemas CRISPR-Cas , Fibroblastos/metabolismo , Fibroblastos/patología , Edición Génica , Técnicas de Inactivación de Genes , Células HEK293 , Células HeLa , Humanos , Metilación , Metiltransferasas/deficiencia , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Conformación de Ácido Nucleico , Fosforilación Oxidativa , Cultivo Primario de Células , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Mitocondrial/metabolismo , ARN de Transferencia/metabolismo , S-Adenosilmetionina/metabolismoRESUMEN
Modified uridine containing taurine, 5-taurinomethyluridine (τm5U), is found at the anticodon first position of mitochondrial (mt-)transfer RNAs (tRNAs). Previously, we reported that τm5U is absent in mt-tRNAs with pathogenic mutations associated with mitochondrial diseases. However, biogenesis and physiological role of τm5U remained elusive. Here, we elucidated τm5U biogenesis by confirming that 5,10-methylene-tetrahydrofolate and taurine are metabolic substrates for τm5U formation catalyzed by MTO1 and GTPBP3. GTPBP3-knockout cells exhibited respiratory defects and reduced mitochondrial translation. Very little τm5U34 was detected in patient's cells with the GTPBP3 mutation, demonstrating that lack of τm5U results in pathological consequences. Taurine starvation resulted in downregulation of τm5U frequency in cultured cells and animal tissues (cat liver and flatfish). Strikingly, 5-carboxymethylaminomethyluridine (cmnm5U), in which the taurine moiety of τm5U is replaced with glycine, was detected in mt-tRNAs from taurine-depleted cells. These results indicate that tRNA modifications are dynamically regulated via sensing of intracellular metabolites under physiological condition.
Asunto(s)
ARN de Transferencia/metabolismo , Taurina/deficiencia , Uridina/análogos & derivados , Animales , Proteínas Portadoras/fisiología , Gatos , Preescolar , Femenino , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/fisiología , Células HEK293 , Células HeLa , Humanos , Mitocondrias/metabolismo , Enfermedades Mitocondriales/genética , ARN de Transferencia/química , Proteínas de Unión al ARN , Uridina/biosíntesisRESUMEN
Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by ptosis, dysphagia, and weakness of proximal limbs. OPMD is caused by the expansion of polyalanine in poly(A)-binding protein, nuclear 1 (PABPN1). Although mitochondrial abnormality has been proposed as the possible etiology, the molecular pathogenesis is still poorly understood. The aim of the study was to specify the mechanism by which expanded PABPN1 causes mitochondrial dysfunction in OPMD. We evaluated whether transgenic mouse model of OPMD, by expressing expanded PABPN1, indeed causes mitochondrial abnormality associated with muscle degeneration. We also investigated the mechanism by which expanded PABPN1 would cause mitochondrial dysfunction in the mouse and cell models of OPMD. Mitochondrial localization of PABPN1 was observed in the muscle fibers of patients with OPMD. Moreover, abnormal accumulation of PABPN1 on the inner membrane of mitochondria and reduced expression of OXPHOS complexes were detected in the muscle fibers of the transgenic mice expressing expanded human PABPN1 with a 13-alanine stretch. In cells expressing PABPN1 with a 10-alanine or 18-alanine stretch, both types of PABPN1 accumulated in the mitochondria and interacted with TIM23 mitochondrial protein import complex, but PABPN1 with 18-alanine stretch decreased the cell viability and aggresome formation. We proposed that the abnormal accumulation of expanded PABPN1 in mitochondria may be associated with mitochondrial abnormality in OPMD.
Asunto(s)
Mitocondrias Musculares/metabolismo , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteína I de Unión a Poli(A)/genética , Proteína I de Unión a Poli(A)/metabolismo , Expansión de Repetición de Trinucleótido , Animales , Estudios de Casos y Controles , Supervivencia Celular , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Mitocondrias Musculares/patología , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Modelos Biológicos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Distrofia Muscular Oculofaríngea/patología , Proteínas Mutantes/química , Fosforilación Oxidativa , Proteína I de Unión a Poli(A)/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Subsets of mitochondrial transfer RNA (tRNA) contain the N6 -isopentenyladenosine (i6 A) or 2-methylthio-N6 -isopentenyladenosine (ms2 i6 A) modification at position A37, which is adjacent to an anticodon. These modifications are essential for efficient protein translation in mitochondria and contribute to energy metabolism. The first step in i6 A and ms2 i6 A modifications is catalyzed by tRNA isopentenyltransferase, which is encoded by the TRIT1 gene. Herein, we report a girl with a developmental delay, frequent episodes of seizures induced by febrile illness, and myoclonic epilepsy who had compound heterozygous missense mutations in TRIT1. A mass spectrometry analysis of RNA nucleoside obtained from the subject's peripheral blood and urine showed a marked decrease in both i6 A and ms2 i6 A modifications. These results suggest that the mitochondrial disorder was caused by defective tRNA isopentenylation arising from a loss-of-function mutation in TRIT1. Furthermore, the present observations suggest that noninvasive biochemical analysis using peripheral blood and urine samples are sufficient for the diagnosis of TRIT1-related disorders, making muscle biopsy for the direct measurement of oxidative phosphorylation unnecessary. Such biochemical analyses before the start of antiepileptic medications would be beneficial to avoid hepatotoxicity in patients with possible mitochondrial disorders.
Asunto(s)
Transferasas Alquil y Aril/genética , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/genética , ARN de Transferencia/genética , Transferasas Alquil y Aril/metabolismo , Alelos , Biomarcadores , Preescolar , Femenino , Genotipo , Humanos , Isopenteniladenosina/metabolismo , ARN de Transferencia/metabolismo , ARN de Transferencia/orinaRESUMEN
The primary cilium is a plasma membrane-protruding sensory organelle that undergoes regulated assembly and resorption. While the assembly process has been studied extensively, the cellular machinery that governs ciliary resorption is less well understood. Previous studies showed that the ciliary pocket membrane is an actin-rich, endocytosis-active periciliary subdomain. Furthermore, Tctex-1, originally identified as a cytoplasmic dynein light chain, has a dynein-independent role in ciliary resorption upon phosphorylation at Thr94. Here, we show that the remodeling and endocytosis of the ciliary pocket membrane are accelerated during ciliary resorption. This process depends on phospho(T94)Tctex-1, actin, and dynamin. Mechanistically, Tctex-1 physically and functionally interacts with the actin dynamics regulators annexin A2, Arp2/3 complex, and Cdc42. Phospho(T94)Tctex-1 is required for Cdc42 activation before the onset of ciliary resorption. Moreover, inhibiting clathrin-dependent endocytosis or suppressing Rab5GTPase on early endosomes effectively abrogates ciliary resorption. Taken together with the epistasis functional assays, our results support a model in which phospho(T94)Tctex-1-regulated actin polymerization and periciliary endocytosis play an active role in orchestrating the initial phase of ciliary resorption.
Asunto(s)
Actinas/fisiología , Cilios/fisiología , Dineínas/metabolismo , Línea Celular , Clatrina/fisiología , Dinaminas , Dineínas/genética , Endocitosis , Células Epiteliales , Humanos , Fosforilación , Multimerización de Proteína , Retina/citologíaRESUMEN
CDK5 regulatory subunit associated protein 1-like 1 (CDKAL1) is a tRNA-modifying enzyme that catalyzes 2-methylthiolation (ms2) and has been implicated in the development of type 2 diabetes (T2D). CDKAL1-mediated ms2 is important for efficient protein translation and regulates insulin biosynthesis in pancreatic cells. Interestingly, an association between T2D and release of growth hormone (GH) has been reported in humans. However, it is unknown whether CDKAL1 is important for hormone production in the pituitary gland. The present study investigated the role of CDKAL1 in GH-producing pituitary adenomas (GHPAs). CDKAL1 activity was suppressed in GHPAs, as evidenced by a decrease in ms2, compared with non-functioning pituitary adenomas (NFPAs), which do not produce specific hormones. Downregulation of Cdkal1 using small interfering and short hairpin RNAs increased the biosynthesis and secretion of GH in rat GH3 cells. Depletion of Cdkal1 increased the cytosolic calcium level via downregulation of DnaJ heat shock protein family (Hsp40) member C10 (Dnajc10), which is an endoplasmic reticulum protein related to calcium homeostasis. This stimulated transcription of GH via upregulation of Pit-1. Moreover, CDKAL1 activity was highly sensitive to proteostatic stress and was upregulated by suppression of this stress. Taken together, these results suggest that dysregulation of CDKAL1 is involved in the pathogenesis of GHPAs, and that modulation of the proteostatic stress response might control CDKAL1 activity and facilitate treatment of GHPAs.
Asunto(s)
Adenoma/genética , Hormona del Crecimiento/biosíntesis , Neoplasias Hipofisarias/genética , ARNt Metiltransferasas/fisiología , Adenoma/metabolismo , Adenoma/patología , Animales , Células Cultivadas , Estrés del Retículo Endoplásmico/fisiología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hormona del Crecimiento/genética , Adenoma Hipofisario Secretor de Hormona del Crecimiento/genética , Adenoma Hipofisario Secretor de Hormona del Crecimiento/metabolismo , Adenoma Hipofisario Secretor de Hormona del Crecimiento/patología , Hormona de Crecimiento Humana/biosíntesis , Hormona de Crecimiento Humana/genética , Humanos , Neoplasias Hipofisarias/metabolismo , Neoplasias Hipofisarias/patología , ARN Interferente Pequeño/farmacología , Ratas , Respuesta de Proteína Desplegada/fisiología , ARNt Metiltransferasas/genéticaRESUMEN
2-Methylthio-N6-isopentenyl modification of adenosine (ms2i6A) is an evolutionally conserved modification that is found in transfer RNAs (tRNAs). We have recently shown that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) specifically converts i6A to ms2i6A at position A37 of four mitochondrial DNA-encoded tRNAs, and that the modification regulates efficient mitochondrial translation and energy metabolism in mammals. Curiously, a previous study reported that ms2i6A is present abundantly in nuclear-derived RNA species such as microRNAs, but not in tRNA fractions. To fully understand the molecular property of ms2i6A, the existence of non-canonical ms2i6A must be carefully validated. In the present study, we examined ms2i6A in total RNA purified from human and murine ρ0 cells, in which mitochondrial DNA-derived tRNAs were completely depleted. The ms2i6A was not detected in these cells at all. We generated a monoclonal antibody against ms2i6A and examined ms2i6A in murine RNAs using the antibody. The anti-ms2i6A antibody only reacted with the tRNA fractions and not in other RNA species. Furthermore, immunocytochemistry analysis using the antibody showed the predominant localization of ms2i6A in mitochondria and co-localization with the mitochondrial elongation factor Tu. Taken together, we propose that ms2i6A is a mitochondrial tRNA-specific modification and is absent from nuclear-encoded RNA species.
Asunto(s)
Núcleo Celular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Isopenteniladenosina/análogos & derivados , Proteínas del Tejido Nervioso/metabolismo , ARN Nuclear/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Núcleo Celular/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Isopenteniladenosina/inmunología , Isopenteniladenosina/metabolismo , Ratones Noqueados , Microscopía Confocal , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/genética , Interferencia de ARN , ARN Nuclear/genética , ARN de Transferencia/genética , ARN de Transferencia/metabolismoRESUMEN
The 2-methylthio (ms2) modification at A37 of tRNAs is critical for accurate decoding, and contributes to metabolic homeostasis in mammals. However, the regulatory mechanism of ms2 modification remains largely unknown. Here, we report that cysteine hydropersulfide (CysSSH), a newly identified reactive sulfur species, is involved in ms2 modification in cells. The suppression of intracellular CysSSH production rapidly reduced ms2 modification, which was rescued by the application of an exogenous CysSSH donor. Using a unique and stable isotope-labeled CysSSH donor, we show that CysSSH was capable of specifically transferring its reactive sulfur atom to the cysteine residues of ms2-modifying enzymes as well as ms2 modification. Furthermore, the suppression of CysSSH production impaired insulin secretion and caused glucose intolerance in both a pancreatic ß-cell line and mouse model. These results demonstrate that intracellular CysSSH is a novel sulfur source for ms2 modification, and that it contributes to insulin secretion.
Asunto(s)
Cisteína/análogos & derivados , Disulfuros/metabolismo , Insulina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , ARN de Transferencia/metabolismo , Azufre/metabolismo , ARNt Metiltransferasas/metabolismo , Animales , Línea Celular , Cisteína/metabolismo , Radicales Libres , Regulación de la Expresión Génica , Células HeLa , Humanos , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Marcaje Isotópico , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Conformación de Ácido Nucleico , ARN de Transferencia/genética , Compuestos de Sulfhidrilo/metabolismo , ARNt Metiltransferasas/genéticaRESUMEN
Reversible infantile liver failure (RILF) is a unique heritable liver disease characterized by acute liver failure followed by spontaneous recovery at an early stage of life. Genetic mutations in MTU1 have been identified in RILF patients. MTU1 is a mitochondrial enzyme that catalyzes the 2-thiolation of 5-taurinomethyl-2-thiouridine (τm5s2U) found in the anticodon of a subset of mitochondrial tRNAs (mt-tRNAs). Although the genetic basis of RILF is clear, the molecular mechanism that drives the pathogenesis remains elusive. We here generated liver-specific knockout of Mtu1 (Mtu1LKO) mice, which exhibited symptoms of liver injury characterized by hepatic inflammation and elevated levels of plasma lactate and AST. Mechanistically, Mtu1 deficiency resulted in a loss of 2-thiolation in mt-tRNAs, which led to a marked impairment of mitochondrial translation. Consequently, Mtu1LKO mice exhibited severe disruption of mitochondrial membrane integrity and a broad decrease in respiratory complex activities in the hepatocytes. Interestingly, mitochondrial dysfunction induced signaling pathways related to mitochondrial proliferation and the suppression of oxidative stress. The present study demonstrates that Mtu1-dependent 2-thiolation of mt-tRNA is indispensable for mitochondrial translation and that Mtu1 deficiency is a primary cause of RILF. In addition, Mtu1 deficiency is associated with multiple cytoprotective pathways that might prevent catastrophic liver failure and assist in the recovery from liver injury.
RESUMEN
Sirtuin 7 (SIRT7) is an NAD+-dependent deacetylase/deacylase, and is involved in a variety of biological processes relevant to the transcription of rRNA, the DNA damage response, tumorigenesis, and metabolism. SIRT7 mRNA is expressed ubiquitously, including in the brain, but there is no detailed information about the anatomical distribution and functional role of SIRT7 in the brain. Here, we demonstrated that SIRT7 is widely expressed in the mouse brain, including in the cortex, striatum, thalamus, hippocampus, and amygdala. Behavioral examinations revealed that Sirt7 knockout (KO) and control mice showed similar levels of freezing behavior immediately after a fear response, but a significant decrease of freezing behavior at 24 h after fear conditioning was observed in Sirt7 KO mice. Histological analysis revealed that there is no apparent structural abnormality of the amygdala and hippocampus, which are regions involved in fear memory consolidation, in Sirt7 KO mice. Our results indicate that SIRT7 is involved in the consolidation of fear memory.
Asunto(s)
Encéfalo/metabolismo , Condicionamiento Clásico/fisiología , Miedo/fisiología , Consolidación de la Memoria/fisiología , Sirtuinas/metabolismo , Animales , Encéfalo/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Distribución TisularRESUMEN
tRNA-isopentenyl transferases (IPTases) are highly conserved enzymes that form isopentenyl-N(6)-A37 (i6A37) on subsets of tRNAs, enhancing their translation activity. Nuclear-encoded IPTases modify select cytosolic (cy-) and mitochondrial (mt-) tRNAs. Mutation in human IPTase, TRIT1, causes disease phenotypes characteristic of mitochondrial translation deficiency due to mt-tRNA dysfunction. Deletion of the Schizosaccharomyces pombe IPTase (tit1-Δ) causes slow growth in glycerol, as well as in rapamycin, an inhibitor of TOR kinase that maintains metabolic homeostasis. Schizosaccharomyces pombe IPTase modifies three different cy-tRNAs(Ser) as well as cy-tRNA(Tyr), cy-tRNA(Trp), and mt-tRNA(Trp). We show that lower ATP levels in tit1-Δ relative to tit1(+) cells are also more decreased by an inhibitor of oxidative phosphorylation, indicative of mitochondrial dysfunction. Here we asked if the tit1-Δ phenotypes are due to hypomodification of cy-tRNA or mt-tRNA. A cytosol-specific IPTase that modifies cy-tRNA, but not mt-tRNA, fully rescues the tit1-Δ phenotypes. Moreover, overexpression of cy-tRNAs also rescues the phenotypes, and cy-tRNA(Tyr) alone substantially does so. Bioinformatics indicate that cy-tRNA(Tyr) is most limiting for codon demand in tit1-Δ cells and that the cytosolic mRNAs most loaded with Tyr codons encode carbon metabolilizing enzymes, many of which are known to localize to mitochondria. Thus, S. pombe i6A37 hypomodification-associated metabolic deficiency results from hypoactivity of cy-tRNA, mostly tRNA(Tyr), and unlike human TRIT1-deficiency does not impair mitochondrial translation due to mt-tRNA hypomodification. We discuss species-specific aspects of i6A37. Specifically relevant to mitochondria, we show that its hypermodified version, ms2i6A37 (2-methylthiolated), which occurs on certain mammalian mt-tRNAs (but not cy-tRNAs), is not found in yeast.