mRNA stability fine-tunes gene expression in the developing cortex to control neurogenesis
Lucas D. Serdar, Jacob R. Egol, Brad Lackford, Brian D. Bennett, Guang Hu, Debra L. Silver
Abstract
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.
Introduction
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].
Materials and method
Mice
Animal use was approved by the Duke Institutional Animal Care and Use Committee (Protocol #: A060-22-03) and followed ethical guidelines provided by the Duke Division of Laboratory Animal Resources. Mouse lines were previously described: Emx1-Cre JAX stock #005628 [55], Trp53lox/lox JAX stock #008462 [79], Nex-Cre [56], Cnot3lox/lox [33]. Primers used for genotyping are listed in S6 Table. For embryo staging, plug dates were defined as embryonic day (E)0.5 on the morning the plug was identified.
Results
SLAM-seq defines the landscape and features of RNA stability in cortical cells
To characterize the mRNA stability landscape of the developing cortex, we performed thiol(SH)-linked alkylation for metabolic sequencing (SLAM-seq) [36] at 3 different developmental stages. We chose E11.5, E14.5, and E16.5 as representative stages of early, middle, and late neurogenesis, respectively. E11.5 cortices are predominantly composed of RGCs, while E14.5 and E16.5 cortices contain increasing numbers of IPs and neurons, along with non-RGC derived cells. Cortices from E11.5, E14.5, and E16.5 embryos were dissociated into single cell suspensions using 3 independent biological replicates per stage and cultured in vitro using conditions permissive for proliferation [37], and 4-thiouridine (4sU) was added to culture media for 20 h to label nascent RNAs.
Discussion
RNA regulation in the developing cortex is dynamic, with rapid changes in expression across dual axes of time and differentiation. RNA expression levels are determined by complementary rates of synthesis and degradation, but the quantitative contribution of the latter to cortical development is largely unknown. We apply omics analyses and genetic approaches to define how RNA turnover controls cortical development. Our transcriptome-wide survey of RNA half-lives across development reveals an in-depth understanding of the cis factors that contribute to RNA turnover, as well as previously unappreciated relationships between RNA stability and developmental gene expression changes.
Acknowledgments
We thank members of the Silver and Hu labs for helpful discussions and careful reading of the manuscript.
Citation: Serdar LD, Egol JR, Lackford B, Bennett BD, Hu G, Silver DL (2025) mRNA stability fine-tunes gene expression in the developing cortex to control neurogenesis. PLoS Biol 23(2): e3003031. https://doi.org/10.1371/journal.pbio.3003031
Academic Editor: Bassem A. Hassan, ICM, FRANCE
Received: August 10, 2024; Accepted: January 23, 2025; Published: February 6, 2025
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.
Data Availability: Sequencing data has been deposited and made publicly available on GEO under accession numbers GSE281690 (RNA-seq) and GSE281693 (SLAM-seq).
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.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: CC3, cleaved caspase 3; cKO, conditional knockout; CSC, codon stabilization coefficient; dcKO, double conditional knockout; GO, gene ontology; IP, intermediate progenitor; NMD, nonsense-mediated RNA decay; PCA, principal component analysis; RGC, radial glial cell
