Previous studies have revealed the critical roles of N6-methyladenosine (m6A) modification of mRNA in embryonic stem cells (ESCs), but the biological function of m6A in large intergenic noncoding RNA (lincRNA) is unknown. Here, we showed that the internal m6A modification of linc1281 mediates a competing endogenous RNA (ceRNA) model to regulate mouse ESC (mESC) differentiation. We demonstrated that loss of linc1281 compromises mESC differentiation and that m6A is highly enriched within linc1281 transcripts. Linc1281 with RRACU m6A sequence motifs, but not an m6A-deficient mutant, restored the phenotype in linc1281-depleted mESCs. Mechanistic analyses revealed that linc1281 ensures mESC identity by sequestering pluripotency-related let-7 family microRNAs (miRNAs), and this RNA-RNA interaction is m6A dependent. Collectively, these findings elucidated the functional roles of linc1281 and its m6A modification in mESCs and identified a novel RNA regulatory mechanism, providing a basis for further exploration of broad RNA epigenetic regulatory patterns.

Clarifying the regulatory mechanisms of embryonic stem cell (ESC) neural differentiation is helpful not only for understanding neural development but also for obtaining high-quality neural progenitor cells required by stem cell therapy of neurodegenerative diseases. Here, we found that long noncoding RNA 1604 (lncRNA-1604) was highly expressed in cytoplasm during neural differentiation, and knockdown of lncRNA-1604 significantly repressed neural differentiation of mouse ESCs both in vitro and in vivo. Bioinformatics prediction and mechanistic analysis revealed that lncRNA-1604 functioned as a novel competing endogenous RNA of miR-200c and regulated the core transcription factors ZEB1 and ZEB2 during neural differentiation. Furthermore, we also demonstrated the critical role of miR-200c and ZEB1/2 in mouse neural differentiation. Either introduction of miR-200c sponge or overexpression of ZEB1/2 significantly reversed the lncRNA-1604 knockdown-induced repression of mouse ESC neural differentiation. Collectively, these findings not only identified a previously unknown role of lncRNA-1604 and ZEB1/2 but also elucidated a new regulatory lncRNA-1604/miR-200c/ZEB axis in neural differentiation. Stem Cells 2018;36:325-336.

DNA methylation and histone methylation (H3K27me3) have been reported as major barriers to induced pluripotent stem cell (iPSC) generation using four core transcription factors (Oct4, Sox2, Klf4, and c-Myc, termed OSKM). Here, to illustrate the possibility of deriving iPSCs via demethylation, as well as the exact effects of DNA methylation and histone modification on gene expression regulation, we performed RNA sequencing to characterize the transcriptomes of ES cells and iPSCs derived by demethylation with miR-29b or shDnmt3a, and carried out integrated analyses. Results showed that OSKM + miR-29b-iPSC was more close to ES cells than the others, and up-regulated genes typically presented with methylated CpG-dense promoters and H3K27me3-enriched regions. The differentially expressed genes caused by introduction of DNA demethylation during somatic cell reprogramming mainly focus on stem cell associated GO terms and KEGG signaling pathways, which may decrease the tumorigenesis risk of iPSCs. These findings indicated that DNA methylation and histone methylation have synergetic effects on regulating gene expression during iPSC generation, and demethylation by miR-29b is better than shDnmt3a for iPSC quality. Furthermore, integrated analyses are superior for exploration of slight differences as missed by individual analysis.

Embryonic stem cells (ESCs) have self‐renewal and multi‐lineage differentiation potential and perform critical functions in development and biomedicine. Several long noncoding RNAs (lncRNAs) have been reported as key regulators of stem cell pluripotency and differentiation. However, the function and regulatory mechanism of lncRNAs during the initiation of ESC differentiation remains unclear. Here, we found that linc1557 was highly expressed in mouse ESCs and required for the initiation of ESC differentiation. Knockdown of linc1557 increased the expression and phosphorylation levels of signal transducer and activator of transcription 3 (STAT3), a key factor in the leukemia inhibitory factor (LIF)/STAT3 signaling pathway. Furthermore, we found that linc1557 directly bound to Stat3 mRNA and affected its stability. The differentially expressed transcriptome after linc1557 knockdown in ESCs was involved primarily in multicellular organism development and cell differentiation as similar to that after Stat3 knockdown. Moreover, either knockdown of Stat3 or addition of a LIF/STAT3 signaling inhibitor rescued the suppressive effects of linc1557 knockdown on the initiation of mouse ESC differentiation. These findings not only elucidated the critical function of linc1557 in the initiation of mouse ESC differentiation but also clarified that its specific mechanism as directly affecting Stat3 mRNA stability, which enhanced the understanding of the lncRNA‐mediated regulatory mechanism for mRNA stability and key signaling pathways in ESC pluripotency and differentiation.

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