Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq analysis of AS-NMD targets in developing mouse neurons

ABSTRACT: The functional coupling between alternative pre-mRNA splicing (AS) and the mRNA quality control mechanism called nonsense-mediated decay (NMD) has been shown to modulate the expression of numerous genes. Previous studies have identified several examples of this regulation in developing neurons. However, the systems-level effects of AS-NMD in this context have been poorly understood. To this end, we performed a longitudinal analysis of gene expression in mouse embryonic stem cells undergoing induced neuronal differentiation, as well as primary neural cells. To improve the detection of NMD-sensitive splice forms, some of the samples were treated with the translation inhibitor cycloheximide or separated into nuclear and cytoplasmic fractions. Our analyses uncovered hundreds of AS-NMD events with significant potential to regulate their host genes. Notably, these events were significantly overrepresented in developmentally downregulated genes. Particularly strong enrichment was detected for alternative cassette exons that trigger NMD upon their inclusion into mature mRNA. Overall, this study extends our understanding of the molecular mechanisms underlying neuronal differentiation and highlights the importance of post-transcriptional regulation gene expression in orchestrating this intricate process.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Mus musculus

SUBMITTER: Eugene Makeyev

PROVIDER: E-MTAB-13134 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Kabuki Syndrome (KS) is a multisystemic rare disorder, characterized by growth delay, distinctive facial features, intellectual disability, and rarely autism spectrum disorder. This condition is mostly caused by de novo mutations of KMT2D, encoding a catalytic subunit of the COMPASS complex involved in enhancer regulation. KMT2D catalyzes the deposition of histone-3-lysine-4 mono-methyl (H3K4Me1) that marks active and poised enhancers. To assess the impact of KMT2D mutations in the chromatin landscape of KS tissues, we have generated patient-derived induced pluripotent stem cells (iPSC), which we further differentiated into neural crest stem cells (NCSC), mesenchymal stem cells (MSC) and cortical neurons (iN). In addition, we further collected blood samples from 5 additional KS patients. To complete our disease modeling cohort we generated an isogenic KMT2D mutant line from human embryonic stem cells, which we differentiated into neural precursor and mature neurons. Micro-electrode-array (MEA)-based neural network analysis of KS iNs revealed an altered pattern of spontaneous network-bursts in a Kabuki-specific pattern. RNA-seq profiling was performed to relate this aberrant MEA pattern to transcriptional dysregulations, revealing that dysregulated genes were enriched for neuronal functions, such as ion channels, synapse activity, and electrophysiological activity. Here we show that KMT2D haploinsufficiency tends to heavily affect the transcriptome of cortical neurons and differentiated tissues while sparing multipotent states, suggesting that KMT2D has a most prevalent role in terminally differentiated cell and activate transcriptional circuitry unique to each cell type. Moreover, thorough profiling of H3K4Me1 unveiled the almost complete uncoupling between this chromatin mark and the regulatory effects of KMT2D on transcription, which is instead reflected by a defect of H3K27Ac. By integrating RNA-seq with ChIP-seq data we defined TEAD and REST as the master effectors of KMT2D haploinsufficiency. Also, we identified a subset of genes whose regulation is controlled by the balance between KMT2D and EZH2 dosage. Finally, we identified the bona fide direct targets of KMT2D in healthy and KS mature cortical neurons and TEAD2 as the main proxy of KMT2D dysregulation in KS. Overall, our study provides the transcriptional and epigenomic characterization of patient-derived tissues as well as iPSCs and differentiated disease-relevant cell types, as well as the identification of KMT2D direct target in cortical neurons, together with the identification of a neuronal phenotype of the spontaneous electrical activity.

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RNA-seq analysis of AS-NMD targets in developing mouse neurons

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