RESUMO
Poly(ADP-ribosylation) (PARylation) is a post-translational modification mediated by a subset of ADP-ribosyl transferases (ARTs). Although PARylation-inhibition based therapies are considered as an avenue to combat debilitating diseases such as cancer and myopathies, the role of this modification in physiological processes such as cell differentiation remains unclear. Here, we show that Tankyrase1 (TNKS1), a PARylating ART, plays a major role in myogenesis, a vital process known to drive muscle fiber formation and regeneration. Although all bona fide PARPs are expressed in muscle cells, experiments using siRNA-mediated knockdown or pharmacological inhibition show that TNKS1 is the enzyme responsible of catalyzing PARylation during myogenesis. Via this activity, TNKS1 controls the turnover of mRNAs encoding myogenic regulatory factors such as nucleophosmin (NPM) and myogenin. TNKS1 mediates these effects by targeting RNA-binding proteins such as Human Antigen R (HuR). HuR harbors a conserved TNKS-binding motif (TBM), the mutation of which not only prevents the association of HuR with TNKS1 and its PARylation, but also precludes HuR from regulating the turnover of NPM and myogenin mRNAs as well as from promoting myogenesis. Therefore, our data uncover a new role for TNKS1 as a key modulator of RBP-mediated post-transcriptional events required for vital processes such as myogenesis.
Assuntos
Desenvolvimento Muscular , Fibras Musculares Esqueléticas , Miogenina , RNA Mensageiro , Tanquirases , Tanquirases/metabolismo , Tanquirases/genética , Humanos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Desenvolvimento Muscular/genética , Animais , Fibras Musculares Esqueléticas/metabolismo , Camundongos , Miogenina/genética , Miogenina/metabolismo , Nucleofosmina , Proteína Semelhante a ELAV 1/metabolismo , Proteína Semelhante a ELAV 1/genética , Estabilidade de RNA/genética , Poli ADP Ribosilação/genética , Linhagem Celular , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Diferenciação Celular/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Células HEK293RESUMO
mRNA stability is the mechanism by which cells protect transcripts allowing their expression to execute various functions that affect cell metabolism and fate. It is well-established that RNA binding proteins (RBPs) such as HuR use their ability to stabilize mRNA targets to modulate vital processes such as muscle fiber formation (myogenesis). However, the machinery and the mechanisms regulating mRNA stabilization are still elusive. Here, we identified Y-Box binding protein 1 (YB1) as an indispensable HuR binding partner for mRNA stabilization and promotion of myogenesis. Both HuR and YB1 bind to 409 common mRNA targets, 147 of which contain a U-rich consensus motif in their 3' untranslated region (3'UTR) that can also be found in mRNA targets in other cell systems. YB1 and HuR form a heterodimer that associates with the U-rich consensus motif to stabilize key promyogenic mRNAs. The formation of this complex involves a small domain in HuR (227-234) that if mutated prevents HuR from reestablishing myogenesis in siHuR-treated muscle cells. Together our data uncover that YB1 is a key player in HuR-mediated stabilization of pro-myogenic mRNAs and provide the first indication that the mRNA stability mechanism is as complex as other key cellular processes such as mRNA decay and translation.
Assuntos
Proteína Semelhante a ELAV 1 , Fibras Musculares Esqueléticas , Fatores de Transcrição , Regiões 3' não Traduzidas , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Linhagem Celular , Animais , Camundongos , Fatores de Transcrição/metabolismoRESUMO
Debilitating cancer-induced muscle wasting, a syndrome known as cachexia, is lethal. Here we report a posttranscriptional pathway involving the RNA-binding protein HuR as a key player in the onset of this syndrome. Under these conditions, HuR switches its function from a promoter of muscle fiber formation to become an inducer of muscle loss. HuR binds to the STAT3 (signal transducer and activator of transcription 3) mRNA, which encodes one of the main effectors of this condition, promoting its expression both in vitro and in vivo. While HuR does not affect the stability and the cellular movement of this transcript, HuR promotes the translation of the STAT3 mRNA by preventing miR-330 (microRNA 330)-mediated translation inhibition. To achieve this effect, HuR directly binds to a U-rich element in the STAT3 mRNA-3'untranslated region (UTR) located within the vicinity of the miR-330 seed element. Even though the binding sites of HuR and miR-330 do not overlap, the recruitment of either one of them to the STAT3-3'UTR negatively impacts the binding and the function of the other factor. Therefore, together, our data establish the competitive interplay between HuR and miR-330 as a mechanism via which muscle fibers modulate, in part, STAT3 expression to determine their fate in response to promoters of muscle wasting.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , MicroRNAs/metabolismo , Atrofia Muscular/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/metabolismo , Biossíntese de Proteínas , RNA Neoplásico/metabolismo , Fator de Transcrição STAT3/biossíntese , Regiões 3' não Traduzidas , Animais , Proteína Semelhante a ELAV 1/genética , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/genética , Atrofia Muscular/genética , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , RNA Neoplásico/genética , Fator de Transcrição STAT3/genéticaRESUMO
HuR (ElavL1) is one of the main post-transcriptional regulators that determines cell fate. Although the role of HuR in apoptosis is well established, the post-translational modifications that govern this function remain elusive. In this study, we show that PARP1/2-mediated poly(ADP)-ribosylation (PARylation) is instrumental in the pro-apoptotic function of HuR. During apoptosis, a substantial reduction in HuR PARylation is observed. This results in the cytoplasmic accumulation and the cleavage of HuR, both of which are essential events for apoptosis. These effects are mediated by a pADP-ribose-binding motif within the HuR-HNS region (HuR PAR-binding site). Under normal conditions, the association of the HuR PAR-binding site with pADP-ribose is responsible for the nuclear retention of HuR. Mutations within this motif prevent the binding of HuR to its import factor TRN2, leading to its cytoplasmic accumulation and cleavage. Collectively, our findings underscore the role of PARylation in controlling the pro-apoptotic function of HuR, offering insight into the mechanism by which PARP1/2 enzymes regulate cell fate and adaptation to various assaults.
Assuntos
Processamento de Proteína Pós-Traducional , Ribose , Mutação , Diferenciação Celular , Domínios ProteicosRESUMO
The master posttranscriptional regulator HuR promotes muscle fiber formation in cultured muscle cells. However, its impact on muscle physiology and function in vivo is still unclear. Here, we show that muscle-specific HuR knockout (muHuR-KO) mice have high exercise endurance that is associated with enhanced oxygen consumption and carbon dioxide production. muHuR-KO mice exhibit a significant increase in the proportion of oxidative type I fibers in several skeletal muscles. HuR mediates these effects by collaborating with the mRNA decay factor KSRP to destabilize the PGC-1α mRNA. The type I fiber-enriched phenotype of muHuR-KO mice protects against cancer cachexia-induced muscle loss. Therefore, our study uncovers that under normal conditions HuR modulates muscle fiber type specification by promoting the formation of glycolytic type II fibers. We also provide a proof-of-principle that HuR expression can be targeted therapeutically in skeletal muscles to combat cancer-induced muscle wasting.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Neoplasias/complicações , Animais , Linhagem Celular , Linhagem Celular Tumoral , Estudos Transversais , Proteína Semelhante a ELAV 1/genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos KnockoutRESUMO
Activation of AMPK has been associated with pro-atrophic signaling in muscle. However, AMPK also has anti-inflammatory effects, suggesting that in cachexia, a syndrome of inflammatory-driven muscle wasting, AMPK activation could be beneficial. Here we show that the AMPK agonist AICAR suppresses IFNγ/TNFα-induced atrophy, while the mitochondrial inhibitor metformin does not. IFNγ/TNFα impair mitochondrial oxidative respiration in myotubes and promote a metabolic shift to aerobic glycolysis, similarly to metformin. In contrast, AICAR partially restored metabolic function. The effects of AICAR were prevented by the AMPK inhibitor Compound C and were reproduced with A-769662, a specific AMPK activator. AICAR and A-769662 co-treatment was found to be synergistic, suggesting that the anti-cachectic effects of these drugs are mediated through AMPK activation. AICAR spared muscle mass in mouse models of cancer and LPS induced atrophy. Together, our findings suggest a dual function for AMPK during inflammation-driven atrophy, wherein it can play a protective role when activated exogenously early in disease progression, but may contribute to anabolic suppression and atrophy when activated later through mitochondrial dysfunction and subsequent metabolic stress.