Abstract
Background7-Methylguanosine (m7G) is one of the most conserved modifications in nucleosides within tRNAs and rRNAs. It plays essential roles in the regulation of mRNA export, splicing, and translation. Recent studies highlighted the importance of METTL1-mediated m7G tRNA methylome in the self-renewal of mouse embryonic stem cells (mESCs) through its ability to regulate mRNA translation. However, the exact mechanisms by which METTL1 regulates pluripotency and differentiation in human induced pluripotent stem cells (hiPSCs) remain unknown. In this study, we evaluated the functions and underlying molecular mechanisms of METTL1 in regulating hiPSC self-renewal and differentiation in vivo and in vitro.MethodsBy establishing METTL1 knockdown (KD) hiPSCs, gene expression profiling was performed by RNA sequencing followed by pathway analyses. Anti-m7G northwestern assay was used to identify m7G modifications in tRNAs and mRNAs. Polysome profiling was used to assess the translation efficiency of the major pluripotent transcription factors. Moreover, the in vitro and in vivo differentiation capacities of METTL1-KD hiPSCs were assessed in embryoid body (EB) formation and teratoma formation assays.ResultsMETTL1 silencing resulted in alterations in the global m7G profile in hiPSCs and reduced the translational efficiency of stem cell marker genes. METTL1-KD hiPSCs exhibited reduced pluripotency with slower cell cycling. Moreover, METTL1 silencing accelerates hiPSC differentiation into EBs and promotes the expression of mesoderm-related genes. Similarly, METTL1 knockdown enhances teratoma formation and mesoderm differentiation in vivo by promoting cell proliferation and angiogenesis in nude mice.ConclusionOur findings provided novel insight into the critical role of METTL1-mediated m7G modification in the regulation of hiPSC pluripotency and differentiation, as well as its potential roles in vascular development and the treatment of vascular diseases.
Highlights
Somatic cells can be transformed into induced pluripotent stem cells functionally equivalent to embryonic stem cells (ESCs) by simultaneously introducing four transcription factors (TFs) Oct4, Sox2, Klf4, and c-Myc [1,2,3]
Gene expression profiling in METTL1 knockdown human induced pluripotent stem cells (hiPSCs) To study the function of the tRNA m7G methyltransferase METTL1 in hiPSCs, METTL1 expression was silenced in hiPSCs using lentivirus expressing METTL1targeting shRNAs (METTL1-KD)
In the upregulated pathways enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG), we found that it is mainly related to immune responses, including systemic lupus erythematosus, graft-versus-host disease, and antigen processing presentation (Fig. S1c). qRT-PCR was performed to assess the expression of 5 representative genes differentially expressed following METTL1 silencing, which confirmed the reproducibility of our RNA sequencing (RNA-seq) findings (Fig. 1c, d)
Summary
Somatic cells can be transformed into induced pluripotent stem cells (iPSCs) functionally equivalent to embryonic stem cells (ESCs) by simultaneously introducing four transcription factors (TFs) Oct, Sox, Klf, and c-Myc [1,2,3]. Recent studies have led to the identification of additional TFs and microRNAs (miRNAs) that can affect the pluripotency and self-renewal capacity of iPSCs [6, 7]. Several studies have indicated that epigenetic modifications regulate stem cell fate decision and development [8,9,10]. The role of post-transcriptional modifications of RNA, including 7-methylguanosine (m7G), in the regulation of hiPSCs fate remains unclear. Our previous study found that METTL1-mediated m7G tRNA modification regulates mRNA translation and the self-renewal, pluripotency, and neural lineage differentiation in mouse embryonic stem cells (mESCs) [13]. We hypothesize that METTL1 may play an important role in hiPSC pluripotency and differentiation
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