RAI1 safeguards fidelity and tempo of human neurodevelopmental gene expression

Human brain development proceeds on an unusually long timeline relative to other species, a feature that is thought to foster advanced cognitive abilities. The Retinoic Acid Induced 1 (RAI1) gene encodes a nucleosome-binding protein, and its haploinsufficiency is responsible for Smith–Magenis Syndrome (SMS), a neurodevelopmental disorder characterized by cognitive impairment with autistic features. However, the role of RAI1 in human neurodevelopment remains unexplored experimentally. Here, we generated isogenic heterozygous and homozygous RAI1 loss-of-function human embryonic stem cell lines and interrogated the roles of RAI1 in neurodevelopmental gene regulation. A longitudinal transcriptome analysis during in vitro cortical development revealed that RAI1 deficiency accelerates the progression of developmental gene expression. Single-cell RNA-seq analysis revealed that RAI1- deficient neuroprogenitors acquire a transient mesoderm-like gene expression signature, followed by a pro-neuronal maturation signature in postmitotic neurons. Unexpectedly, the developmental acceleration signature was exacerbated during NGN2-induced excitatory neuron differentiation, isolating the roles of RAI1 in neuronal differentiation from non-neuronal functions. Together, these results identify RAI1 as a suppressor of the mesodermal lineage program and as a brake that slows the tempo of human neurodevelopmental gene expression.