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Multiple knockout mouse models indicate a role of miR-124a in neuronal maturation

ABSTRACT: MicroRNA-124a (miR-124a) is one of the most abundantly expressed miroRNAs in the central nervous system (CNS) and encoded by three genomic loci of miR-124a-1/2/3 in mammals; however, its in vivo roles and mechanisms in neuronal development and function remain ambiguous. In the present study, we investigated the effect of loss of miR-124a on neuronal differentiation in mice and embryonic stem (ES) cells. Since primary miR-124a-3 shows a background level expression in the brain and we could not obtain miR-124a-1/2/3 triple knockout (TKO) mice by mating, we generated and analyzed miR-124a-1/2 double knockout (DKO) mice. We found that miR-124a-1/2 DKO mice exhibit perinatal lethality. RNA-sequencing analysis demonstrated that the expression levels of proneural and neuronal marker genes are almost unchanged between the control and miR-124a-1/2 DKO brains, while the genes related to neuronal synaptic formation and function were enriched in the down-regulated genes of the miR-124a-1/2 DKO brain. In addition, the transcription regulator Tardbp/TDP-43, whose loss leads to defects in neuronal maturation and function, was inactivated in the miR-124a-1/2 DKO brain. We next generated miR-124a-1/2/3 TKO ES cells by using the CRISPR-Cas9 system as an alternative to miR-124a-1/2/3 TKO mice. Phase-contrast microscopic, immunocytochemical, and gene expression analyses showed that miR-124a-1/2/3 TKO ES cell lines can differentiate into neurons. Collectively, these results suggest that miR-124a plays roles in neuronal maturation rather than neurogenesis in vivo, and may advance our understanding of functional roles of microRNAs in CNS development in vivo.

ORGANISM(S): Mus musculus

PROVIDER: GSE196356 | GEO | 2022/07/07

REPOSITORIES: GEO

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Project description:MicroRNAs (miRNAs) play roles in diverse developmental and disease processes. Distinct miRNAs have hundreds to thousands of conserved mRNA binding sites, but typically direct only modest repression via single sites. Cotargeting of individual mRNAs by different miRNAs could potentially achieve stronger and more complex patterns of repression. Comparing target sets of different miRNAs, we identified hundreds of pairs of miRNAs that share more mRNA targets than expected (often by two-fold or more) relative to stringent controls, with particularly high levels of cotargeting among brain-enriched miRNAs. We profiled miRNA expression changes over a cell culture model of neuronal differentiation, and selected the non-homologous cotargeting miRNA pair miR-138 and miR-137 to study functionally. We generated a CRISPR-mediated mir-138 double knockout (DKO) cell line in the murine CAD cell culture model of neuronal differentiation. mir-138 DKO cells lacking both mir-138 loci were unable to differentiate and project neurites following serum withdrawal. Further characterization of the DKO cells by RNA-seq showed that loss of miR-138 resulted in a de-differentiated state compared to WT cells. Specificity of the mir-138 DKO was confirmed by the rescue of neurite growth upon addition of a miR-138 mimic. Interestingly, addition of a miR-137 mimic, but not mimics of other neuronal miRNAs, could also rescue the neurite growth phenotype and allow mir-138 DKO cells to differentiate, and transcriptome profiling revealed similar gene expression changes. Clustering of all cotargeting pairs revealed a group of 9 predominantly brain-enriched miRNAs that share many targets. In reporter assays, subsets of these miRNAs together repressed gene expression by 5- to 10-fold, often exhibiting cooperative repression. Together, our results uncover an unexpected pattern in which combinations of miRNAs collaborate to robustly repress cotargets, and suggest important developmental roles for cotargeting.

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Multiple knockout mouse models indicate a role of miR-124a in neuronal maturation

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