YTHDC1 recognizes METTL16-dependent m⁶A on caRNAs and coordinates cotranscriptional splicing.

Abstract

<i>N⁶</i>-methyladenosine (m⁶A) RNA modification regulates diverse biological process. The m⁶A writers and downstream readers collaboratively undertake m⁶A-mediated RNA metabolism, yet the functional specificity among different writers and readers remains poorly understood. Using limb organogenesis as a development model, we uncover a critical and specific functional axis between the m⁶A reader YTHDC1 and writer METTL16. Depletion of either YTHDC1 or METTL16-but not METTL3-causes severe limb malformations, revealing unexpected functional selectivity. Mechanistically, we demonstrate that YTHDC1 specifically recognizes METTL16-deposited m⁶A marks on chromatin-associated RNAs, orchestrating cotranscriptional splicing of genes vital for cell cycle progression and DNA repair. Loss of YTHDC1 triggers genome-wide transcription arrest and dysregulates key developmental gene expression programs. Importantly, chromatin-bound YTHDC1 recruits splicing factors to transcriptional complex through liquid-liquid phase separation (LLPS), with alkalic arginine residues in its C-terminal region being molecular determinants. Our findings identified a selective and specific METTL16-m⁶A-YTHDC1 axis that couples RNA modification with cotranscriptional splicing during mammalian organogenesis, providing molecular insights into how epitranscriptomic regulation governs developmental decisions.