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mRNA stability fine-tunes gene expression in the developing cortex to control neurogenesis [1]

['Lucas D. Serdar', 'Department Of Molecular Genetics', 'Microbiology', 'Duke University Medical Center', 'Durham', 'North Carolina', 'United States Of America', 'Jacob R. Egol', 'Brad Lackford', 'National Institute Of Environmental Health Sciences']

Date: 2025-02 

RNA abundance is controlled by rates of synthesis and degradation. Although mis-regulation of RNA turnover is linked to neurodevelopmental disorders, how it contributes to cortical development is largely unknown. Here, we discover the landscape of RNA stability regulation in the cerebral cortex and demonstrate that intact RNA decay machinery is essential for corticogenesis in vivo. We use SLAM-seq to measure RNA half-lives transcriptome-wide across multiple stages of cortical development. Leveraging these data, we discover cis-acting features associated with RNA stability and probe the relationship between RNA half-life and developmental expression changes. Notably, RNAs that are up-regulated across development tend to be more stable, while down-regulated RNAs are less stable. Using compound mouse genetics, we discover CNOT3, a core component of the CCR4-NOT deadenylase complex linked to neurodevelopmental disease, is essential for cortical development. Conditional knockout of Cnot3 in neural progenitors and their progeny in the developing mouse cortex leads to severe microcephaly due to altered cell fate and p53-dependent apoptosis. Finally, we define the molecular targets of CNOT3, revealing it controls expression of poorly expressed, non-optimal mRNAs in the cortex, including cell cycle-related transcripts. Collectively, our findings demonstrate that fine-tuned control of RNA turnover is crucial for brain development.

Funding: This work was supported by the Extramural and Intramural Research Programs of the National Institutes of Health: F32HD107972 to L.D.S., R01NS083897, R01NS120667, R37NS110388, R01MH132089, R21NS128374 to D.L.S., and Z01ES102745 to G.H. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Here, we employ multi-pronged approaches to understand the principles and functions of RNA degradation in the developing brain ( Fig 1A ). We use SLAM-seq to globally measure RNA half-lives across multiple stages of mouse development. From this, we discover key features governing RNA stability in the cortex and uncover relationships between RNA turnover rates and gene expression changes across development. We then employ Cnot3 conditional knockout mouse models to discover cellular and molecular mechanisms by which RNA decay machinery controls cortical neurogenesis. Our findings reveal the importance of RNA stability control in the developing cortex and establish the CCR4-NOT complex as a key factor contributing to proper gene expression and cellular function during corticogenesis.

A key regulator of mRNA stability is the CCR4-NOT deadenylase complex [ 20 ]. This multi-subunit complex catalyzes the removal of 3′ poly(A) tails from mRNAs, typically one of the first steps in mRNA degradation. CNOT3 is a non-enzymatic component of the CCR4-NOT complex and required for efficient deadenylation and mRNA degradation [ 21 – 23 ]. Mutations in CNOT3 as well as another component, CNOT1, are linked to neurodevelopmental pathologies characterized by a spectrum of intellectual disability, speech delay, seizures, and behavioral problems [ 24 – 30 ]. Of note, CNOT3 is categorized as a “High-confidence, syndromic” autism risk gene in the SFARI autism gene database. Previous work implicates CNOT3 in diverse stem cell populations, including cardiomyocytes [ 31 , 32 ], embryonic stem cells [ 33 , 34 ], and spermatogonial stem cells [ 35 ]. Despite clinical significance of CNOT3 in neurodevelopment, its molecular function in cortical development is entirely unknown.

In line with diverse cellular processes, the transcriptomes of progenitors and neurons are dynamic during corticogenesis [ 4 – 7 ]. These developmental gene expression changes are controlled by transcriptional and posttranscriptional mechanisms [ 8 , 9 ]. RNA stability regulation is emerging as an important modulator of gene expression during cortical development. Previous work has revealed roles for core components of the RNA decay machinery, including the exosome [ 10 ] and the nonsense-mediated RNA decay (NMD) pathway [ 11 – 16 ]. Several studies have also highlighted the importance of additional cis- and trans-acting regulators of stability in cortical development. For example, the oscillatory RNA expression pattern of the Notch signaling pathway gene Hes1 in cortical progenitors is maintained in part by microRNA-mediated degradation [ 17 , 18 ]. In addition, the abundant epitranscriptomic mark N6-methyladenosine (m 6 A) destabilizes RNA in cortical cells and influences neurogenesis [ 19 ]. While these studies highlight fundamental roles of mRNA stability in cortical development, our understanding of this process is limited. Indeed, a survey of RNA half-lives across development is lacking, as is a mechanistic understanding of how RNA turnover impacts the developing brain.

(A) Overview of cortical development showing RGCs generating neurons directly, or indirectly through IPs, with goals of this study schematized below. (B) Approach for SLAM-seq experiment, with relative cellular composition of the cortex at the 3 developmental stages used in this study (n = 3 biological replicates per stage). (C) Histograms showing distribution of RNA half-lives. (D, E) Venn diagrams for top 10% most and least stable transcripts identified at each stage. (F) CSC values for each of the 61 non-stop codons. (G) Half-lives of RNAs binned into quartiles based on percentage of optimal (positive CSC) codons. (H) Half-lives of RNAs with or without m 6 A. *** p < 0.001. One-way ANOVA with Tukey’s HSD post hoc test (J), Wilcoxon rank-sum test (K). Underlying data for this figure can be found in S1 Data . CSC, codon stabilization coefficient; IP, intermediate progenitor; RGC, radial glial cell.

The cerebral cortex is essential for higher order functions, including cognitive reasoning, and somatosensory, motor, and visual processing. These processes rely on proper embryonic development, and defective embryonic corticogenesis can lead to neurodevelopmental disorders, including autism, schizophrenia, and intellectual disability. The developmental trajectory and underlying biological and molecular events necessary to construct the cortex during development are generally well defined [ 1 , 2 ]. In mice, the main neural progenitors are radial glial cells (RGCs), which produce neurons directly, and intermediate progenitors (IPs), which are derived from RGCs and are also neurogenic ( Fig 1A ). The mature mammalian cortex consists of a stereotypical six-layered architecture that forms in an inside-out fashion. Neurons born early in development (~embryonic day (E)12.5 –E13.5 in mouse) occupy deep layers, while late born neurons (~E14.5 –E16.5) typically occupy superficial layers [ 3 ]. Thus, corticogenesis is a dynamic process in which progenitor potency and neuronal composition changes with development.

Results

Conditional knockout of Cnot3 leads to microcephaly and disrupted neuronal lamination Our data thus far indicate that RNA stability regulation is a key feature of cortical development, implicated in gene expression changes across stages. Our data also indicate that codon optimality is highly correlated with mRNA stability in the embryonic cortex (Fig 1G). Mechanistically, codon content and its effects on translation are sensed by the CCR4-NOT complex to drive degradation of non-optimal mRNAs [54]. This led us to investigate the in vivo functional consequences of mis-regulating the CCR4-NOT complex during cortical development. To address this, we focused on CNOT3, a central component of the CCR4-NOT deadenylase complex (Fig 4A). CNOT3 is essential for deadenylation [21–23] and is linked to neurodevelopmental pathologies [24–28], making it a strong candidate for investigating the role of the complex in cortical development. Importantly, inspection of scRNA-seq data sets of the developing mouse cortex revealed broad Cnot3 expression in both progenitors and neurons [7]; however, its function in these cell types is unknown. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Cnot3 controls cerebral cortex size and neuronal number. (A) Schematic of CCR4-NOT complex with CNOT3 indicated in red. (B) cKO strategy using Emx1-Cre to target RGCs and their progeny, including IPs and neurons. (C) Western blot using whole cortex protein lysates from E12.5 control (ctrl), conditional heterozygous (cHet), or cKO embryos. Quantifications shown to the right (n = 4 embryos per genotype). (D) Whole mount images and coronal cross sections of E18.5 brains. Quantifications of cortex area and thickness shown to the right (n = 4 embryos per genotype). (E–H) Immunofluorescence for indicated neuronal markers in E18.5 cortical sections, with quantifications shown to the right. n = 3–5 embryos per genotype. *p < 0.05, **p < 0.01, ***p < 0.001. One-way ANOVA with Tukey’s HSD post hoc test. Error bars represent standard deviation. Scale bars: 1 mm (D, top), 500 μm (D, bottom), 50 μm (E–H). Underlying data for this figure can be found in S4 Data. cKO, conditional knockout; IP, intermediate progenitor; RGC, radial glial cell. https://doi.org/10.1371/journal.pbio.3003031.g004 We generated conditional knockouts (cKOs) of Cnot3 in RGCs and their progeny by crossing Cnot3lox/lox mice with Emx1-Cre [55] (Fig 4B). At E12.5 (approximately 3 days after the onset of Cre-mediated recombination), we observed an 84% and 82% reduction in CNOT3 protein and RNA expression, respectively, in cKO cortices (Figs 4C and S3A). Interestingly, expression of CNOT1 was also reduced. Reduction of CNOT1 protein likely occurs via translational and/or posttranslational mechanisms, as we observed no significant change in Cnot1 RNA measured by RT-qPCR (S3A Fig). No statistically significant changes were observed for CNOT2 expression at either the protein or RNA levels. As CNOT1 is essential for scaffolding the complex, this suggests that the integrity of the entire CCR4-NOT complex may be impaired in this mouse model. These data demonstrate the Cnot3 cKO can be faithfully used to assess roles of this complex in cortical development. To determine the requirement of Cnot3 for cortical development, we first examined brains at E18.5, when neurogenesis is largely complete. cKO mice exhibited profound microcephaly, with a 38% reduction in cortical area, and a similar 38% reduction in cortical thickness (Fig 4D). In contrast, conditional heterozygous (cHet) brain sizes were comparable to control. This indicates that CNOT3 is required for overall cortical size. The cortex forms a six-layered laminar architecture, with early born neurons present in deep layers and later born neurons primarily present in superficial layers [3]. To examine the extent to which Cnot3 ablation affects glutamatergic projection neurons, we performed immunostaining for TBR1 (Layer VI), CTIP2 (Layer V), RORβ (Layer IV), and LHX2 (Layer II/III). In cKO brains compared to control, we observed a ~28% reduction in TBR1+ neuron density, a near complete loss of CTIP2+ and RORβ+ neurons, and a 65% reduction in LHX2+ neurons (Fig 4E–4H). cHet brains were similar to control. In addition to reduced density, neuronal distribution for some markers was also disrupted in cKO brains (S3B–S3E Fig). These data indicate that CNOT3 is essential for number and laminar position of glutamatergic neurons produced throughout cortical development.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3003031

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