Isoform-Specific Splicing of ANK2 by PTBP2 Orchestrates Retinal Pigment Epithelial-to-Neuron Fate Conversion

While transcription factor-based approaches have been extensively studied, the role of post-transcriptional regulation, particularly alternative splicing (AS), in neuronal fate acquisition remains poorly defined. Here, we demonstrate that the concurrent knockdown of the splicing regulator PTBP2 and the barrier protein p53 synergistically enhances the neuronal conversion of human retinal pigment epithelial (hRPE-19) cells when combined with ASCL1 and MiR-9/9*-124 (AMnp).

Transcriptomic and splicing analyses reveal that PTBP2 depletion induces widespread AS changes, most notably promoting near-complete inclusion of exon 36 in the ANK2 gene, which encodes a key regulator of axon initial segment assembly. Functional assays confirm that the loss of exon 36 significantly impairs neuronal induction, establishing ANK2 isoform switching as a mechanistic requirement for reprogramming. Moreover, AMnp-reprogrammed neurons display upregulation of photoreceptor markers (RHO, L/M-opsin, and recoverin) and transcriptional signatures of epigenetic remodeling, suggesting that chromatin memory may interact with AS to influence subtype identity.