The N6-methyladenosine (m6A) RNA modification plays essential roles in multiple biological processes, including stem cell fate determination. To explore the role of the m6A modification in pluripotent reprogramming, we used RNA-seq to map m6A effectors in human iPSCs, fibroblasts, and H9 ESCs, as well as in mouse ESCs and fibroblasts. By integrating the human and mouse RNA-seq data, we found that 19 m6A effectors were significantly upregulated in reprogramming. Notably, IGF2BPs, particularly IGF2BP1, were among the most upregulated genes in pluripotent cells, while YTHDF3 had high levels of expression in fibroblasts. Using quantitative PCR and Western blot, we validated the pluripotency-associated elevation of IGF2BPs. Knockdown of IGF2BP1 induced the downregulation of stemness genes and exit from pluripotency. Proteome analysis of cells collected at both the beginning and terminal states of the reprogramming process revealed that the IGF2BP1 protein was positively correlated with stemness markers SOX2 and OCT4. The eCLIP-seq target analysis showed that IGF2BP1 interacted with the coding sequence (CDS) and 3’UTR regions of the SOX2 transcripts, in agreement with the location of m6A modifications. This study identifies IGF2BP1 as a vital pluripotency-associated m6A effector, providing new insight into the interplay between m6A epigenetic modifications and pluripotent reprogramming.

中文翻译：

---

分析 m6A 效应子在多能重编程调节中的作用

N6-甲基腺苷 (m6A) RNA 修饰在多种生物过程中发挥着重要作用，包括干细胞命运决定。为了探索 m6A 修饰在多能重编程中的作用，我们使用 RNA-seq 绘制了人 iPSC、成纤维细胞和 H9 ESC 以及小鼠 ESC 和成纤维细胞中的 m6A 效应子图谱。通过整合人类和小鼠 RNA-seq 数据，我们发现 19 个 m6A 效应子在重编程中显着上调。值得注意的是，IGF2BP，特别是 IGF2BP1，是多能细胞中上调最多的基因之一，而 YTHDF3 在成纤维细胞中表达水平较高。使用定量 PCR 和蛋白质印迹，我们验证了 IGF2BP 的多能性相关升高。 IGF2BP1 的敲除诱导干性基因下调并退出多能性。对重编程过程开始和结束状态时收集的细胞进行蛋白质组分析，结果显示 IGF2BP1 蛋白与干性标记 SOX2 和 OCT4 呈正相关。 eCLIP-seq 目标分析显示 IGF2BP1 与 SOX2 转录本的编码序列 (CDS) 和 3'UTR 区域相互作用，与 m6A 修饰的位置一致。这项研究将 IGF2BP1 确定为重要的多能相关 m6A 效应子，为 m6A 表观遗传修饰和多能重编程之间的相互作用提供了新的见解。