Transcriptomic Analysis of the m6A Reader YTHDF2 in the Maintenance and Differentiation of Human Embryonic Stem Cells

As the most abundant internal modification on mRNAs, N6-methyladenosine (m6A) has been discovered to be involved in different biological processes. Mostly determined by m6A methyl-transferases (m6A writers) and demethylases (m6A erasers), different cell types possess differential m6A profiles of transcriptomes. However, the interpretation of the m6A-encoded epitranscriptomic information needs m6A readers to bind and recruit different machinery for regulating the target mRNAs, which in turn, may regulate cell fates. The functions of the m6A readers in the regulation of the maintenance and differentiation of human embryonic stem cells (hESCs) remain largely unknown. In this study, we deleted the whole genomic region of the m6A reader YTHDF2, and discovered that YTHDF2 is dispensible for the maintenance, but probably important for the differention of hESCs, especially for the differentiation towares ectoderm. Furthermore, we identified ROBO1 as a potential target gene by DF2 in regulating hESC to neuroectoderm differentiation. This study reveals the potential roles of the m6A reader DF2 in regulating the specification of neuroectodermal cell fate. Overall design: hESCs (WT_hESCs and hESCs_DF2-KO) were cultured in Nuwacell® ncTarget hPSC Medium (Nuwacell, RP01020) on Matrigel-coated plates. Endoderm (WT_endoderm and endoderm_DF2-KO) and mesoderm (WT_mesoderm and mesoderm_DF2-KO) cells were induced from hESCs by culturing in STEMdiff™ Definitive Endoderm Medium (STEMCELL, 05110) or STEMdiff™ Definitive Mesoderm Medium (STEMCELL, 05220) for 4 days. Similarly, STEMdiffTM Neural induction medium supplemented with SMADi (STEMCELL, 8581) were used for induction and culture of ectoderm (WT_ectoderm and ectoderm_DF2-KO) cells, but a longer induction of around 13 days was applied. hESCs with ADAR expression (hESC_ADAR and hESC_DF2-ADAR) were induced with doxcycyline for 24 hours before its RNA got extracted.