Human stem cell-derived models provide the promise of accelerating our understanding of brain disorders. But, not knowing whether they possess the ability to mature beyond late mid-fetal stages potentially limits their utility. So, we leveraged a directed differentiation protocol to comprehensively assess maturation in vitro . Based on genome-wide analysis of the epigenetic clock, transcriptomics, as well as RNA-editing, we observe that 3D human cortical organoids reach postnatal stages between 250–300 days, a timeline paralleling in vivo development. We demonstrate the presence of several known developmental milestones, including switches in the histone deacetylase complex and NMDA receptor subunits, which we confirm at the protein and physiological levels. These results suggest that important components of an intrinsic in vivo developmental program persist in vitro . We further map neurodevelopmental and neurodegenerative disease risk genes onto in vitro gene expression trajectories to provide a resource and webtool (GECO) to guide disease modeling.
Transcriptomic analyses of postmortem brains have begun to elucidate the molecular abnormalities in autism spectrum disorder (ASD). However, a crucial pathway involved in synaptic development, RNA editing, has not yet been studied on a genome-wide scale. Here, we profiled global patterns of adenosine-to-inosine (A-to-I) editing in a large cohort of post-mortem ASD brains. We observed a global bias for hypoediting in ASD brains, which was shared across brain regions and involved many synaptic genes. We show that the Fragile X proteins, FMRP and FXR1P, interact with ADAR proteins and modulate A-to-I editing. Furthermore, we observed convergent patterns of RNA editing alterations in ASD and Fragile X syndrome, establishing this as a molecular link between these related diseases. Our findings, which are corroborated across multiple datasets, including dup15q cases associated with intellectual disability, highlight RNA editing dysregulation in ASD and reveal novel mechanisms underlying this disorder.
Adenosine-to-inosine (A-to-I) editing, mediated by the ADAR enzymes, diversifies the transcriptome by altering RNA sequences. Recent studies reported global changes in RNA editing in disease and development. Such widespread editing variations necessitate an improved understanding of the regulatory mechanisms of RNA editing. Here, we study the roles of >200 RNA-binding proteins (RBPs) in mediating RNA editing in two human cell lines. Using RNA-sequencing and global protein-RNA binding data, we identify a number of RBPs as key regulators of A-to-I editing. These RBPs, such as TDP-43, DROSHA, NF45/90 and Ro60, mediate editing through various mechanisms including regulation of ADAR1 expression, interaction with ADAR1, and binding to Alu elements. We highlight that editing regulation by Ro60 is consistent with the global up-regulation of RNA editing in systemic lupus erythematosus. Additionally, most key editing regulators act in a cell type-specific manner. Together, our work provides insights for the regulatory mechanisms of RNA editing.
In eukaryotes, nascent RNA transcripts undergo an intricate series of RNA processing steps to achieve mRNA maturation. RNA editing and alternative splicing are two major RNA processing steps that can introduce significant modifications to the final gene products. By tackling these processes in isolation, recent studies have enabled substantial progress in understanding their global RNA targets and regulatory pathways. However, the interplay between individual steps of RNA processing, an essential aspect of gene regulation, remains poorly understood. By sequencing the RNA of different subcellular fractions, we examined the timing of adenosine-to-inosine (A-to-I) RNA editing and its impact on alternative splicing. We observed that >95% A-to-I RNA editing events occurred in the chromatin-associated RNA prior to polyadenylation. We report about 500 editing sites in the 3 ′ acceptor sequences that can alter splicing of the associated exons. These exons are highly conserved during evolution and reside in genes with important cellular function. Furthermore, we identified a second class of exons whose splicing is likely modulated by RNA secondary structures that are recognized by the RNA editing machinery. The genome-wide analyses, supported by experimental validations, revealed remarkable interplay between RNA editing and splicing and expanded the repertoire of functional RNA editing sites.
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