Investigating the role of A-to-I RNA editing by ADARs in corticogenesis using human cerebral organoid models

About This Grant

ABSTRACT The development of the cerebral cortex is one of the most intricate processes in neurobiology. Disruptions to this complex and highly regulated process are central to neurodevelopmental disorders (NDDs), which collectively impact an estimated 317 million individuals globally. Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by the ADAR (adenosine deaminase acting on RNA) family, has emerged as a potent regulator of post-transcriptional gene regulation in the brain. A-to-I editing is highly dynamic in the developing human brain and has been implicated in a range of NDDs, including autism, schizophrenia, and epilepsy. A growing body of evidence suggests editing may contribute to neuronal maturation, synaptic regulation, and the diversification of the brain transcriptome. Yet, a single-cell resolution map of A-to-I editing and direct evidence of essential ADAR function during human corticogenesis have never been achieved. This proposal presents a comprehensive and mechanistically focused investigation into how editing shapes transcriptional and translational landscapes during human cortical development. It represents the most comprehensive and detailed investigation of A-to-I RNA editing in the developing human brain to date (Aim 1). I propose to map the RNA editome at unprecedented scale: across over 2.3 million cells from both fetal brain tissue from 26 individual donors and human cortical organoids. This work will reveal cell type- and lineage-specific editing programs and evaluate the fidelity of organoids in modeling A-to-I editing dynamics. This work will be achieved without cell sorting or complex tissue pre-processing prior to sequencing with MARINE, a first-in-class computational tool for detecting editing that preserves single-cell resolution. Additionally, this proposal is the first systematic dissection of ADAR enzyme function in a complex human model of corticogenesis (Aim 2). To systematically evaluate the importance of the ADARs in corticogenesis, each ADAR is repressed in several cell lines engineered for CRISPR interference and ribosome-based translational profiling (Ribo-STAMP). ADAR-repressed cortical organoids are evaluated with several modalities to understand how ADAR shapes cell fate specification, lineage progression, and mRNA translation. The use of Ribo-STAMP provides the first transcriptome-wide readout of ADAR-dependent translation in the developing human cortex, revealing regulatory layers inaccessible by transcriptional profiling alone. By uniting high-resolution transcriptomic, translational, and morphological profiling in tractable human models, this study establishes a systems-level framework for decoding post-transcriptional regulation in the developing brain and lays critical groundwork for therapeutic advances in NDDs.