Epitranscriptomic variation in banded newts (Ommatotriton vittatus) across life stages and sexes in the semi-arid habitat in northern Israel

The banded newt, Ommatotriton vittatus, is considered endangered in Israel, which is the southern border of its distribution and requires adaptation to highly unstable habitats. Banded newts have both aquatic and terrestrial forms, going through metamorphosis and various phenotypic changes during their life. Our group has previously reported on the extensive transcriptomic remodeling based on sex and especially the life stage of the newt. These dramatic changes in gene expression are likely accompanied by extensive epitranscriptomic regulation, which remains poorly understood. In this study, direct RNA sequencing was used to characterize m⁶A RNA modifications in 12 newts from the Nehalit population. We focused on 127 genes with >30 differentially modified regions (DMRs, with a differential modification score > 5) in their transcripts, involved in structural, translational, and extracellular matrix processes. Among them, 18 genes showed clustering of methylation patterns according to life stage, particularly keratins and translation-related proteins, suggesting that m⁶A plays a regulatory role in structural remodeling and developmental transitions. In contrast, the majority of DMR genes were associated with housekeeping and stress-response functions and did not show life-stage-specific clustering. Cross-analysis with differential expression data further indicated that muscle, immune, and connective tissue pathways are co-regulated at both the transcriptional and epitranscriptomic levels. These findings provide the first evidence of m⁶A methylation patterns in O. vittatus and highlight their role in developmental transitions. The results advance understanding of transcriptomic-epitranscriptomic regulation in amphibian plasticity, and more generally, in vertebrate development.