Long-read proteogenomic atlas of human neuronal differentiation reveals isoform diversity informing neurodevelopmental risk mechanisms.

Reference annotations incompletely capture the transcript diversity of the human brain, obscuring the genetic architecture of neurodevelopmental disorders. Here, we integrated deep long-read RNA sequencing and proteomics in iPSC-derived cortical neurons to generate a high-resolution proteogenomic atlas of human neuron development. We identify 182,371 mRNA isoforms (over half novel) and provide direct peptide evidence for the translation of hundreds of novel protein-coding sequences. Crucially, population genetics demonstrate that novel splice sites are under strong negative selection, comparable to known missense mutations, confirming their functional importance. During neuronal maturation, we observe that ASD risk genes undergo dynamic isoform switching, including microexon usage and intron retention, that remodels key protein domains and regulatory regions. Furthermore, we uncover widespread, long-range coordination between splicing and polyadenylation, revealing complex regulatory logic. Critically, our atlas enables variant reinterpretation in ASD, demonstrating that an isoform-centric view is essential for interpreting pathogenic variation in neurodevelopment.