EFTUD2 Regulates Cortical Morphogenesis via Modulation of Caspase‐3 and Aifm1 Splicing Pathways

Abstract Elongation Factor Tu GTP‐Binding Domain Containing 2 (EFTUD2), a core spliceosomal GTPase associated with Mandibulofacial Dysostosis with Microcephaly (MFDM), plays a mechanistically undefined role in cerebral development. To investigate its pathophysiological contributions, murine models are generated through conditional Eftud2 ablation and in utero electroporation of human pathogenic EFTUD2 variants into cortical neural stem cells (NSCs). Embryonic NSC‐specific Eftud2 knockout resulted in cortical disorganization and microcephaly, while pathogenic variants led to significant neuronal loss. Integrative transcriptomic and immunofluorescence analyses revealed that Eftud2 deficiency triggers apoptotic pathways, contributing to cortical malformations. Mechanistic studies using RNA co‐immunoprecipitation and full‐length transcriptome sequencing demonstrated that Eftud2 directly interacts with Caspase3 and Aifm1 transcripts, regulating their alternative splicing to generate pro‐apoptotic isoforms. Splicing assays functionally validated this regulatory mechanism, showing its role in activating cell death pathways and disrupting neurodevelopmental homeostasis. These findings elucidate EFTUD2's critical role in maintaining apoptotic balance during corticogenesis and identify defective splicing regulation as the molecular basis of MFDM. This study provides insights for advancing diagnostic frameworks and therapeutic strategies for neurodevelopmental disorders.