RESUMO
Myogenesis is a highly orchestrated process whereby muscle precursor cells, myoblasts, develop into muscle fibers to form skeletal muscle during embryogenesis and regenerate adult muscle. Here, we studied the RNA-binding protein FUS (fused in sarcoma), which has been implicated in muscular and neuromuscular pathologies but is poorly characterized in myogenesis. Given that FUS levels declined in human and mouse models of skeletal myogenesis, and that silencing FUS enhanced myogenesis, we hypothesized that FUS might be a repressor of myogenic differentiation. Interestingly, overexpression of FUS delayed myogenesis, accompanied by slower production of muscle differentiation markers. To identify the mechanisms through which FUS inhibits myogenesis, we uncovered RNA targets of FUS by ribonucleoprotein immunoprecipitation (RIP) followed by RNA-sequencing (RNA-seq) analysis. Stringent selection of the bound transcripts uncovered Tnnt1 mRNA, encoding troponin T1 (TNNT1), as a major effector of FUS influence on myogenesis. We found that in myoblasts, FUS retained Tnnt1 mRNA in the nucleus, preventing TNNT1 expression; however, reduction of FUS during myogenesis or by silencing FUS released Tnnt1 mRNA for export to the cytoplasm, enabling TNNT1 translation and promoting myogenesis. We propose that FUS inhibits myogenesis by suppressing TNNT1 expression through a mechanism of nuclear Tnnt1 mRNA retention.
RESUMO
RNA modifications, including N6-methyladenosine (m6A), critically modulate protein expression programs in a range of cellular processes. Although the transcriptomes of cells undergoing senescence are strongly regulated, the landscape and impact of m6A modifications during senescence are poorly understood. Here, we report a robust m6A modification of PTCHD4 mRNA, encoding Patched Domain-Containing Protein 4, in senescent cells. The METTL3/METTL14 complex was found to incorporate the m6A modification on PTCHD4 mRNA; addition of m6A rendered PTCHD4 mRNA more stable and increased PTCHD4 production. MeRIP RT-qPCR and eCLIP analyses were used to map this m6A modification to the last exon of PTCHD4 mRNA. Further investigation identified IGF2BP1, but not other m6A readers, as responsible for the stabilization and increased abundance of m6A-modified PTCHD4 mRNA. Silencing PTCHD4, a transmembrane protein, enhanced growth arrest and DNA damage in pre-senescent cells and sensitized them to senolysis and apoptosis. Our results indicate that m6A modification of PTCHD4 mRNA increases the production of PTCHD4, a protein associated with senescent cell survival, supporting the notion that regulating m6A modification on specific mRNAs could be exploited to eliminate senescent cells for therapeutic benefit.
Assuntos
Adenosina , Sobrevivência Celular , Senescência Celular , Metiltransferases , RNA Mensageiro , Proteínas de Ligação a RNA , Humanos , Senescência Celular/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Metiltransferases/metabolismo , Metiltransferases/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Sobrevivência Celular/genética , Apoptose/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dano ao DNARESUMO
An intriguing effect of short-term caloric restriction (CR) is the expansion of certain stem cell populations, including muscle stem cells (satellite cells), which facilitate an accelerated regenerative program after injury. Here, we utilized the MetRSL274G (MetRS) transgenic mouse to identify liver-secreted plasminogen as a candidate for regulating satellite cell expansion during short-term CR. Knockdown of circulating plasminogen prevents satellite cell expansion during short-term CR. Furthermore, loss of the plasminogen receptor KT (Plg-RKT) is also sufficient to prevent CR-related satellite cell expansion, consistent with direct signaling of plasminogen through the plasminogen receptor Plg-RKT/ERK kinase to promote proliferation of satellite cells. Importantly, we are able to replicate many of these findings in human participants from the CALERIE trial. Our results demonstrate that CR enhances liver protein secretion of plasminogen, which signals directly to the muscle satellite cell through Plg-RKT to promote proliferation and subsequent muscle resilience during CR.