Abstract: Objective To systematically characterize N⁶-methyladenosine (m⁶A) modifications on mitochondrial messenger RNA (mt-mRNA), define its features, and investigate its changes during neurodevelopment and in brain tumors.Methods Thirteen sequencing datasets generated by seven m⁶A profiling techniques were integrated to compare m⁶A distribution and relative methylation levels across chromosomes and to delineate an m⁶A modification landscape of mt-mRNA. In addition, mouse cerebral cortices and cerebella at different developmental stages, together with three brain tumor cell lines, were collected, and representative transcripts were validated by methylated RNA immunoprecipitation followed by quantitative polymerase chain reaction (MeRIP-qPCR).Results m⁶A modifications on mt-mRNA were detected across multiple human, mouse and rat tissues and cell types, albeit at levels lower than those of nuclear-encoded transcripts. During mouse cortical and cerebellar development, mt-mRNA m⁶A levels showed stage-dependent changes. Compared with normal brain tissues, mt-mRNA m⁶A levels were decreased in glioblastoma and medulloblastoma. Conclusions This study confirms the presence of m⁶A modifications in the mitochondrial transcriptome and demonstrates their dynamic regulation during neurodevelopment and in the pathogenesis of brain tumors,providing a new perspective for understanding mitochondrial function and its involvement in diseases.

Key words: mitochondrial mRNA, N⁶-methyladenosine, neurodevelopment, glioblastoma, medulloblastoma