MBNL1-dependent alternative splicing of NUMA1 shapes neuronal identity during fibroblast-to-neuron reprogramming

Direct neuronal reprogramming provides a powerful strategy to generate neurons from somatic cells without passing through a pluripotent state. However, the post-transcriptional mechanisms that refine neuronal identity downstream of fate induction remain poorly understood. Here, we identify alternative splicing as a critical regulator of neuronal identity during fibroblast-to-neuron reprogramming. Knockdown of the RNA-binding protein MBNL1 establishes a distinct reprogramming state (AMmnp) that enhances neuronal maturation and strongly biases induced neurons toward an inhibitory, GABAergic identity, without markedly altering overall conversion efficiency. Transcriptomic analyses revealed coordinated repression of extracellular matrix-associated gene programs together with widespread remodeling of alternative splicing. Among MBNL1-dependent splicing events, NUMA1 exon 16 emerged as a prominent target. Functional manipulation of NUMA1 isoforms demonstrated that exon 16 inclusion suppresses neuronal marker expression specifically in the AMmnp context, whereas exon skipping is permissive for neuronal differentiation. These findings establish MBNL1-dependent alternative splicing of NUMA1 exon 16 as a post-transcriptional mechanism that refines neuronal identity acquisition during direct reprogramming, highlighting alternative splicing as an active determinant of induced neuron quality and subtype specification.