RESUMO
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
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.