pADP-ribosylation regulates the cytoplasmic localization, cleavage, and pro-apoptotic function of HuR.

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.

Tankyrase-1 regulates RBP-mediated mRNA turnover to promote muscle fiber formation.

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.

The formation of HuR/YB1 complex is required for the stabilization of target mRNA to promote myogenesis.

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.

Autophagy and heat-shock response impair stress granule assembly during cellular senescence.

Stress granules (SGs) are membraneless organelles formed in response to insult. These granules are related to pathological granules found in age-related neurogenerative diseases such as Parkinson's and Alzheimer's. Previously, we demonstrated that senescent cells, which accumulate with age, exposed to chronic oxidative stress, are unable to form SGs. Here, we show that the senescent cells' inability to form SGs correlates with an upregulation in both the heat-shock response and autophagy pathways, both of which are well-established promoters of SG disassembly. Our data also reveals that the knockdown of HSP70 and ATG5, important components of the heat-shock response and autophagy pathways, respectively, restores the number of SGs formed in senescent cells exposed to chronic oxidative stress. Surprisingly, under these conditions, the depletion of HSP70 or ATG5 did not affect the clearance of these SGs during their recovery from chronic stress. These data reveal that senescent cells possess a unique heat-shock and autophagy-dependent ability to impair the formation of SGs in response to chronic stress, thereby expanding the existing understanding of SG dynamics in senescent cells and their potential contribution to age-related neurodegenerative diseases.

Stress granules counteract senescence by sequestration of PAI-1.

Cellular senescence is a physiological response by which an organism halts the proliferation of potentially harmful and damaged cells. However, the accumulation of senescent cells over time can become deleterious leading to diseases and physiological decline. Our data reveal a novel interplay between senescence and the stress response that affects both the progression of senescence and the behavior of senescent cells. We show that constitutive exposure to stress induces the formation of stress granules (SGs) in proliferative and presenescent cells, but not in fully senescent cells. Stress granule assembly alone is sufficient to decrease the number of senescent cells without affecting the expression of bona fide senescence markers. SG-mediated inhibition of senescence is associated with the recruitment of the plasminogen activator inhibitor-1 (PAI-1), a known promoter of senescence, to these entities. PAI-1 localization to SGs increases the translocation of cyclin D1 to the nucleus, promotes RB phosphorylation, and maintains a proliferative, non-senescent state. Together, our data indicate that SGs may be targets of intervention to modulate senescence in order to impair or prevent its deleterious effects.

Destabilization of nucleophosmin mRNA by the HuR/KSRP complex is required for muscle fibre formation.

HuR promotes myogenesis by stabilizing the MyoD, myogenin and p21 mRNAs during the fusion of muscle cells to form myotubes. Here we show that HuR, via a novel mRNA destabilizing activity, promotes the early steps of myogenesis by reducing the expression of the cell cycle promoter nucleophosmin (NPM). Depletion of HuR stabilizes the NPM mRNA, increases NPM protein levels and inhibits myogenesis, while its overexpression elicits the opposite effects. NPM mRNA destabilization involves the association of HuR with the decay factor KSRP as well as the ribonuclease PARN and the exosome. The C terminus of HuR mediates the formation of the HuR-KSRP complex and is sufficient for maintaining a low level of the NPM mRNA as well as promoting the commitment of muscle cells to myogenesis. We therefore propose a model whereby the downregulation of the NPM mRNA, mediated by HuR, KSRP and its associated ribonucleases, is required for proper myogenesis.

HuR and miR-1192 regulate myogenesis by modulating the translation of HMGB1 mRNA.

Upon muscle injury, the high mobility group box 1 (HMGB1) protein is upregulated and secreted to initiate reparative responses. Here we show that HMGB1 controls myogenesis both in vitro and in vivo during development and after adult muscle injury. HMGB1 expression in muscle cells is regulated at the translational level: the miRNA miR-1192 inhibits HMGB1 translation and the RNA-binding protein HuR promotes it. HuR binds to a cis-element, HuR binding sites (HuRBS), located in the 3'UTR of the HMGB1 transcript, and at the same time miR-1192 is recruited to an adjacent seed element. The binding of HuR to the HuRBS prevents the recruitment of Argonaute 2 (Ago2), overriding miR-1192-mediated translation inhibition. Depleting HuR reduces myoblast fusion and silencing miR-1192 re-establishes the fusion potential of HuR-depleted cells. We propose that HuR promotes the commitment of myoblasts to myogenesis by enhancing the translation of HMGB1 and suppressing the translation inhibition mediated by miR-1192.

The translation inhibitor pateamine A prevents cachexia-induced muscle wasting in mice.

Cachexia, or muscle-wasting syndrome, is one of the major causes of death in patients affected by diseases such as cancer, AIDS and sepsis. However, no effective anti-cachectic treatment is currently available. Here we show that a low dose of pateamine A, an inhibitor of translation initiation, prevents muscle wasting caused by the cytokines interferon γ and tumour necrosis factor α or by C26-adenocarcinoma tumours. Surprisingly, although high doses of pateamine A abrogate general translation, low doses selectively inhibit the expression of pro-cachectic factors such as inducible nitric oxide synthase. This selectivity depends on the 5'UTR of inducible nitric oxide synthase messenger RNA (mRNA) that, unlike the 5'UTR of MyoD mRNA, promotes the recruitment of inducible nitric oxide synthase mRNA to stress granules, where its translation is repressed. Collectively, our data provide a proof of principle that nontoxic doses of compounds such as pateamine A could be used as novel drugs to combat cachexia-induced muscle wasting.

Oxidative Stress Increases the Number of Stress Granules in Senescent Cells and Triggers a Rapid Decrease in p21waf1/cip1 Translation.

Very little is known as to how the accumulation of senescent cells during aging may affect our ability to cope with various stresses. Here we show that the assembly of stress granules (SGs) is part of the early events used by senescent cells to respond to certain stresses. Although SGs can form in response to stress during senescence activation, their number significantly increases once the cells are fully senescent. This increase correlates with a rapid decrease in the expression levels of the cyclin kinase inhibitor p21, an important activator of senescence. Throughout stress, p21 mRNA is stabilized and localizes to SGs, but only during late senescence does this localization interferes with its translation. Additionally, we observed that when the stress is relieved, senescent cells produce lower levels of p21 protein, which correlates with a small delay in SG disassembly. Therefore, our data suggest that SG formation and the reduction in p21 protein levels represent two main events by which senescent cells respond to stress.