| N~6-methyladenosine(m~6A)represents the most prevalent modification observed in eukaryotic messenger RNA(m RNA),repetitive RNA,and long non-coding RNA.This modification plays a pivotal role in regulating RNA stability and gene expression.Notably,during the maternal-to-zygotic transition in early embryos and throughout zygotic genome activation(ZGA),m~6A modification is crucial for maintaining maternal RNA stability and facilitating the degradation of ZGA transcripts.The activation of ZGA is essential for embryonic totipotency,and the inhibition of m~6A on ZGA-associated transcripts can impede the timely degradation of these transcripts,thereby adversely affecting embryonic development.2C-like cells(2-cell-like cells),a subset of embryonic stem cells(ESCs),exhibit characteristics akin to totipotent stem cells.These cells express ZGA-related genes and retroposons characteristic of totipotent 2-cell embryos,and their developmental potential surpasses that of typical ESCs.Despite their significance,the scarcity of 2C-like cells has limited extensive research.The role of m~6A modification in the transition from pluripotent stem cells to 2C-like cells remains poorly understood,and it is unclear whether the stabilization of2C-like cells can be achieved by modulating m~6A modifications or their associated proteins.In this study,we employed a 2C-like cell reporter system to isolate 2C-like cells from ESCs via flow cytometry.We then characterized the transcriptome m~6A modification profile of 2C-like cells using an ultralow-input m~6A RNA immunoprecipitation followed by sequencing technology(ULI-Me RIP-seq),which was previously developed in our laboratory.This profile was compared with those of ESCs and two-cell embryos.Our findings revealed that 2C-like cells share some RNA m~6A modification distribution characteristics with two-cell embryos while retaining numerous characteristics similar to ESCs.Differential gene expression analysis between 2C-like cells and ESCs indicated that genes with increased m~6A modifications exhibited significant upregulation,whereas downregulated transcripts showed no significant correlation with m~6A changes.Further analysis revealed that m~6A modifications were significantly enriched on ZGA transcripts compared to non-ZGA transcripts,suggesting selective enrichment of m~6A on ZGA transcripts associated with high levels of gene expression.Additionally,m~6A modifications were enriched on the repetitive sequence MERVL,accompanied by high expression levels of MERVL,mirroring the patterns observed in two-cell embryos.Single-cell RNA sequencing(sc RNA-seq)analysis revealed that transcripts bearing m~6A modifications were degraded significantly faster than their unmodified counterparts during the transition from the 2C-like state.Further investigations indicated that zygotic genome activation(ZGA)transcripts with m~6A modifications,including Zscan4c,Zfp352,Usp17lc,and the repetitive sequence MERVL,were downregulated at a significantly higher rate compared to ZGA transcripts lacking m~6A modifications.Although ZGA transcripts with m~6A modifications also exhibited a faster downregulation rate compared to non-ZGA transcripts,the difference was less pronounced than that observed within ZGA transcripts.These findings suggest that m~6A modifications may play a role in the targeted degradation of ZGA transcripts and MERVL,facilitating their downregulation upon exit from the 2C-like state.The application of STM2457,an inhibitor of the methyltransferase METTL3,resulted in a significant reduction of m~6A modifications within embryonic stem cells(ESCs),yet it did not markedly alter the proportion of 2C-like cells within the ESC population.Totipotency withdrawal experiments demonstrated that inhibition of METTL3 significantly delayed the downregulation of m~6A-modified ZGA transcripts,with similar outcomes observed following the knockdown of METTL3 using short hairpin RNA(sh RNA).Bioinformatics analysis confirmed that m~6A modifications on ZGA transcripts and MERVL were mediated by METTL3.Subsequent RNA degradation assays indicated that inhibition of METTL3 significantly impeded the degradation of ZGA transcripts.Moreover,comparative transcriptomic analysis between 2C-like cells and ESCs revealed a significant reduction in the expression of the m~6A reader protein IGF2BP2 in 2C-like cells.Inhibition of IGF2BP2 using the inhibitor CW1-2 in ESCs markedly reduced cell proliferation rates.Flow cytometry analysis demonstrated that inhibition of IGF2BP2 significantly increased the proportion of 2C-like cells within the ESC population.Gene expression analysis indicated that inhibition of IGF2BP2 significantly elevated the expression of ZGA genes and MERVL,while concurrently decreasing the expression of pluripotency genes.RNA immunoprecipitation sequencing(RIP-seq)revealed that many transcripts downregulated during the transition from ESCs to 2C-like cells were bound to IGF2BP2,such as the pluripotency factor Klf2 with m~6A modifications,suggesting that IGF2BP2 may influence the transformation of ESCs into 2C-like cells by interacting with m~6A-modified pluripotent transcripts.This study elucidated the transcriptome m~6A modification profile of 2C-like cells,enhancing our understanding of the role of m~6A modifications in the transition between 2C-like cells and ESCs.The key findings of this study are as follows:(1)The distribution of m~6A modifications during the state transition between ESCs and 2C-like cells was highly conserved and not extensively reprogrammed.(2)RNA m~6A modifications in 2C-like cells are highly enriched in highly expressed ZGA transcripts.(3)During the exit from the 2C-like state,RNA m~6A modifications established by METTL3 promoted the silencing of ZGA genes and MERVL.(4)Inhibition of the m~6A reader protein IGF2BP2 significantly increased the number of 2C-like cells.Overall,this study revealed the role of m~6A modification in the exit from the 2C-like state and identified a factor that can promote the transition of ESCs to 2C-like cells,providing new insights for the stabilization of 2C-like cell cultures. |
