Direct RNA sequencing reveals multi-layered regulation of the aging transcriptome

Abstract BACKGROUND: Aging involves complex molecular alterations across tissues; however, a comprehensive understanding of epitranscriptomic dynamics remains elusive. RESULTS: Here, leveraging high-quality dRNA-seq, we simultaneously constructed a multi-tissue (lung, skeletal muscle, heart, and hippocampus), cross-species (mouse and human) dynamics atlas of RNA features during aging, namely N 6-methyladenosine (m 6 A), 5-methylcytidine (m 5 C), pseudouridine (Ψ), inosine, alternative polyadenylation (APA), and splicing. Our analysis revealed widespread non-linear, age-dependent shifts across these RNA features, exhibiting both conserved patterns and striking tissue specificity. Strikingly, a pronounced, transient peak in these shift events of RNA features occurred at mid-life (12 months of age in mice, 46 years of age in human), and affected the expression of aging-related, mitochondrial, and metabolic genes. CONCLUSIONS: Collectively, we present a high-resolution atlas of m 6 A, m 5 C, pseudouridine and inosine modifications, alternative splicing and poly(A) site usage across tissues and aging stages, derived from direct RNA sequencing. This resource offers an unprecedented foundation for dissecting the RNA-centric mechanisms that govern mammalian aging.