RNA m6A-binding protein YTHDF2 regulates neoblast proliferation in planarian regeneration by targeting ASTACIN Signaling

Introduction: Planarians exhibit remarkable notable regenerative capacity, through the self-renewal of the neoblast stem cells. The RNA N⁶-methyladenosine (m⁶A)-binding protein YTHDF2 recognizes m⁶A modified regions on the mRNA and facilitates their degradation, which is critical in RNA metabolism and tumorigenesis. However, its precise function and underlying mechanism in regulating neoblasts during planarian regeneration remain unclear.

Objectives: This study was aimed at investigating the role of DjYTHDF2.2 in regulating neoblast self-renewal and regeneration in planarians and identifying its functional targets and underlying mechanisms.

Methods: The spatiotemporal expression of Djythdf2.2 was analyzed via quantitative reverse-transcription PCR and whole-mount in situ hybridization. Djythdf2.2 function in tissue homeostasis and regeneration was assessed through RNA interference (RNAi) and immunostaining. MeRIP-seq and RNA-seq were used to identify m⁶A-modified targets, followed by spatial co-localization analysis. MeRIP-qPCR and actinomycin D assays were utilized to examine the association between the Djastacin (Djast) mRNA and YTHDF2. Finally, rescue experiments were performed using double RNAi.

Results: Djythdf2.2 showed pronounced upregulation after amputation. Its knockdown severely impaired tissue homeostasis and regeneration by suppressing neoblast proliferation. Integrated methylated RNA immunoprecipitation Sequencing (MeRIP-seq) and RNA-seq identified the metalloendopeptidase gene Djastacin (Djast) as a key m⁶A-modified target of DjYTHDF2.2. Djythdf2.2 knockdown resulted in the accumulation of Djast transcripts. Furthermore, staining revealed that these Djast transcripts co-localized with the Djythdf2.2 and H3P-positive neoblasts, respectively. Notably, Djast knockdown substantially restored regeneration and neoblast self-renewal in Djythdf2.2-deficient Animals. In addition, actinomycin D assays showed that the half-life of Djast mRNA was dramatically increased upon Djythdf2.2 knockdown.

Conclusion: These findings show that DjYTHDF2.2 promotes neoblast self-renewal and regeneration by targeting Djast mRNA for degradation, which is a novel m⁶A-dependent regulatory axis that is essential for stem cell maintenance and tissue regeneration in planarians. This study highlights the conserved role of YTHDF2-mediated post-transcriptional control in stem Cell Biology and regenerative mechanisms.

AST; M(6)A; Neoblasts; Planarian regeneration; YTHDF2.