Increasing evidences demonstrated that m6A mRNA modification are involved in numerous biological functions in senescence.In this study, we found that m6A RNA modifications is heavily involved in senescence of SkMSCs. Initially, both reduction of m6A modification and METTL3 expression were observed in aged SkMSCs. Then, we found that downregulation of METTL3 lead to m6A modifications downregulation and SOCS3 upregulation, accelerating SkMSC senescence. Subsequently, upregulation of METTL3 enhanced m6A modifications and inhibited senescence by the JAK2-STAT3 signaling pathway through upregulation of SOCS3 in SkMSCs. Finally, IGF2BP1 recognized and stabilized m6A-modified SOCS3 mRNA to relieve senescence of SkMSC. Collectively, our results suggest that upregulation of METTL3 and m6A modifications can relieve senescence of SkMSCs via IGF2BP1 modulating enhancement of SOCS3 mRNA stability, which disturbed the JAK2-STAT3 signaling pathway. These results not only identify the roles of m6A and
METTL3 in senescence of SkMSC, and also implied the possibility to develop therapeutic strategies for aging-related muscle disorders.
By targeting different mRNAs in young and aged SkMSCs, m6A regulation by METTL3 is involved in regulating biological processes in very different way. Aberrant expression of m6A are known to support critical germinal center (GC) functions, cell malignant transformation and carcinogenesis through regulating the expression of targeted mRNAs18–20. Remarkably, abnormal m6A modifications are reported to regulate senescence, which may be implicated in age-related disorders. Moreover, dysregulation of m6A was also reported to be involved in age-related neural activity and Alzheimer's disease 21,22. These findings imply a regulatory function of m6A in aging. Consistent with our findings, recent studies reported that METTL3 regulates m6A levels in myoblasts and in the transition of myoblasts to different cell states12. Decreased m6A modifications has been reported to regulate the fate of bone marrow mesenchymal stem cells and the bone-fat balance during skeletal aging. However, the earliest stages of terminal differentiation commitment were not investigated and the upstream signal that leads to the decline in METTL3 transcription has not yet in full shape. Our study further employed young and aged SkMSCs and found m6A loss in aged SkMSCs due to reduction of METTL3. Moreover, knockout of METTL3 was observed to accelerate senescence in SkMSCs, implying METTL3 as well as m6A play important roles in the senescence of SkMSCs. Similar to our study, Li also reported that METTL3 deficiency shortens the lifespan in Drosophila23.
In aged SkMSCs, m6A profiling observed the declined methylation and protein coding pathways. Protein coding is a general stereotypes in aging. As a protein coding regulator, SOCS3 can directly interact with JAK2, suppressing the JAK2/STAT3 signaling pathway and thus plays roles in regulating cell signaling connectivity under physiological and pathological conditions.24–26. Consistently, reduction of SOCS3expression in cells causes abnormal epigenetic and metabolic reprogramming27,28. Recent studies reported that decreased m6A methylation results in a reduction of SOCS3 mRNA29–31. However, regulatory mechanism of SOCS3 in senescence still remains uninvestigated. Our study identified the reduction of m6A modification and SOCS3 and its association in aged mouse SkMSCs. METTL3 knockout decreased the stability of SOCS3, may owe to reduced m6A modifications around their stop codons preferentially recognized and stabilized by IGF2BP132–34. Really, numerous studies suggest that IGF2BP1 selectively recognizes and promotes mRNA stability35,36. Our data demonstrated that m6A modification and SOCS3 are involved senescence of SkMSCs, suggesting that intervention of m6A modification and m6A might be a potential strategy for regulating cell senescence.
SOCS3 function as a negative regulator of the JAK-STAT pathway, which plays a crucial role in age-related diseases and cellular aging37,38. The role of SOCS3 in aging regulation is achieved by inhibiting JAK-STAT pathway, and other related mediators. SOCS3can bind to JAK2 and suppress the activation of STAT3, thereby regulating cellular aging. Recent studies have reported that SOCS3 has a detrimental role of inflammation and that reduction of SOCS3 can cause primary microglial dysfunction and the development age-related retinal microgliopathy in mice38. In addition, knock out of SOCS3 in mice can restrain cell proliferation and growth. In a mouse model, METTL3 was reported to modify SOCS3 mRNAs by m6A methylation, which decreased the degradation of SOCS3 and increased its translation, thereby suppressing JAK2/STAT3 signaling and consequently promote cell proliferation and growth39. Moreover, we found that m6A modification are involved in regulating SOCS3 translation in a METTL3/ IGF2BP1-orchestrated manner. Consequently, we demonstrated that METTL3 promotes the m6A methylation of SOCS3 mRNA, and then IGF2BP1 recognizes and stabilizes SOCS3 mRNA and promotes SOCS3 protein expression. Consistently, a recent study have shown that the reduction of phosphorylation of JAK2/STAT3 in aged Klotho deficient (+/-) mice; however, the mechanism by which DR1 affecting the aging of cells has not been reported40. Our study that m6A methylation of SOCS3 regulate the senescence of SkMSCs through JAK2–STAT3 signaling. Epigenetic modification of m6A has a powerful influence on cellular senescence and affects the cell fate and leads to the overexpression of target protein in aged mice to relieve age-related disease; however, further exploration of the molecular mechanisms remain is required 41,42. Our data provides the evidence that SOCS3 binds to JAK2 and promote its degradation.
Several limitations need be noted. Firstly, the role of SOCS3 to METTL3-mediated m6A methylation may vary due to cell type,cellular signal and microenvironment. Second, we have explored the association between m6A modification and SOCS3, however, we failed to identify the exact binding site of SOCS3 that interact with downstream target. Thirdly, Further in vivo study is needed to reveal the role of m6A and manipulation of m6A modification in senescence. Despite these limitations, our data have demonstrated that reduction of m6A is a senescence biomarker in SkMSCs and that the restoration of m6A through METTL3 upregulation can alleviate senescence of SkMSCs.
In summary, our study shows that IGF2BP1-mediated improvement of SOCS3 mRNA stability is enhanced by METTL3-mediated m6A methylation to alleviate the senescence of SkMSCs. The association between SOCS3 and m6A modification implies that the manipulation of m6A modification may serve as a promising treatment for age-related disease. Our present study provided the molecular mechanism of m6A in aging and identifies METLL3 and SOCS3 as therapeutic target
for the diagnosis and treatment of age- associated disorders. Further studies are needed to identify the exact binding site of SOCS3 that interact with JAK2 and we speculate that SOCS3 may have other targets besides JAK2 in SkMSCs.