Characterization Of Rna N~6-Methyladenosine Modification-Related Genes In Xenopus Tropicalis Embryos

N6-methyladenosine(m~6A)modification is the methylation on the sixth nitrogen atom of adenine in RNA.The biological functions of m~6A are mediated by a number of writer(methylase),eraser(demethylase),and reader proteins.m~6A plays important roles in vertebrate embryogenesis.Due to its low abundance(only0.1-0.4%of the total adenosines in mRNA),mouse model suffers from insufficient embryonic materials for molecular mechanism studies of m~6A function.Thus,the true diploid frog Xenopus tropicalis can serve as an ideal model for functional characterization of m~6A in embryogenesis.In this study,m~6A was detected in all the early stage X.tropicalis embryos tested upon dot blot analysis.Whole-mount in situ hybridization analyses indicate that the writer genes mettl3,mettl14,and wtap are mainly expressed in animal pole cells and neural crest cells of early stage X.tropicalis embryos.The expression level of eraser gene fto is very low,while eraser gene alkbh5 is also mainly expressed in animal pole cells and neural crest cells of early stage embryos.The reader genes ythdf1,ythdf2,ythdf3,ythdc1,ythdc2,and hnrnpc are mainly expressed in animal pole cells,neural crest cells,as well as developing eyes of early stage embryos.Western blot analysis did not reveal very differential expression patterns of m~6A reader genes in X.tropicalis early stage embryos.RT-PCR analysis indicates that reader genes ythdf1,ythdf2,ythdf3,ythdc1,and ythdc2 are all broadly expressed in various X.tropicalis adult organs.Together,these data suggest that RNA m~6A modification plays roles in various aspects of embryogenesis in X.tropicalis.ythdf1,ythdf2,ythdf3,ythdc1,and ythdc2 knockout X.tropicalis G0 founders have been generated with a CRISPR/Cas9-mediated targeted gene disruption method.Healthy F1 heterozygotes for ythdf1,ythdf2,and ythdf3 knockout have also been obtained.ythdf3 knockout homozygous embryos obtained by early cold shock gynogenesis with a G0 female founder appear healthy.Taken together,this study provides a solid basis for further systematically exploring the molecular mechanism of m~6A functioning in X.tropicalis embryogenesis.