Abstract:
N6-methyladenosine (m6A) RNA methylation has emerged as a regulator of virtually all RNA functions across the domains of life. Among the numerous important functions of m6A, regulation of pluripotency to balance its maintenance versus resolution has been implicated as one of its critical functions during embryogenesis. However, investigation of the ancestral bias of mRNA methylation in the stemness balance in early Metazoa remains. To this end, the epitranscriptomic landscape of the earlydivergent metazoan Hydra vulgaris was examined along with its physiological modulation. The dynamic and abundant stem cell pool of Hydra supporting easily traceable processes like budding and regeneration along with genetic tractability make it a suitable model system to identify the ancestral bias of m6A in pluripotency regulation. The presence of m6A and its machinery was confirmed in H. vulgaris. Mining of regeneration and single-cell RNA-seq data revealed that m6A machinery and its target transcripts are more highly expressed in differentiating progenitor cell populations than in their parental stem cells. Upon ectopic Wnt activation, m6A marks increased on Wnt signaling and other transcripts, particularly at AT-rich repeats rather than canonical DRACH motifs. Global Wnt activation induced tandem coding sequence hypermethylation on differentially methylated mRNAs without significantly altering transcript abundance. Ablation of m6A led to mRNA hypomethylation and impaired morphallaxis but not budding kinetics, indicating a pro-differentiation role of this mark. Moreover, in vitro assays confirmed strong methyltransferase and demethylase activities of purified HvMettl3/14 and HvAlkbh5 proteins, respectively. These findings suggest that m6A modification originally evolved to promote differentiation, functioning in coordination with Wnt signaling dynamics.
