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Drosha regulates neurogenesis by controlling Neurogenin 2 expression independent of microRNAs

Abstract

Temporal regulation of embryonic neurogenesis is controlled by hypostable transcription factors. The mechanism of the process is unclear. Here we show that the RNase III Drosha and DGCR8 (also known as Pasha), key components of the microRNA (miRNA) microprocessor, have important functions in mouse neurogenesis. Loss of microprocessor in forebrain neural progenitors resulted in a loss of stem cell character and precocious differentiation whereas Dicer deficiency did not. Drosha negatively regulated expression of the transcription factors Neurogenin 2 (Ngn2) and NeuroD1 whereas forced Ngn2 expression phenocopied the loss of Drosha. Neurog2 mRNA contains evolutionarily conserved hairpins with similarities to pri-miRNAs, and associates with the microprocessor in neural progenitors. We uncovered a Drosha-dependent destabilization of Neurog2 mRNAs consistent with microprocessor cleavage at hairpins. Our findings implicate direct and miRNA-independent destabilization of proneural mRNAs by the microprocessor, which facilitates neural stem cell (NSC) maintenance by blocking accumulation of differentiation and determination factors.

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Figure 1: Microprocessor inactivation at E13.5 induces neural-progenitor differentiation and results in a distinct phenotype compared to Dicer1 cKO.
Figure 2: Drosha knockdown reduces S-phase labeling but does not induce cell-cycle exit.
Figure 3: Drosha regulates proneural mRNA levels.
Figure 4: Drosha knockdown induces Ngn2 protein expression and activation of neurogenesis.
Figure 5: Expression of Ngn2 phenocopies Drosha inhibition and microprocessor inactivation and induces NeuroDmCherry expression.
Figure 6: Drosha interacts and destabilizes hairpins within Neurog2 mRNA.

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Acknowledgements

We thank N. Kim (Seoul National University) for Drosha constructs, F. Guillemot (UK National Institute for Medical Research) for Neurog2 and shRNA constructs, and P. Herrare for beta2/NeuroD promoter, members of the Taylor laboratory for helpful discussions, and F. Sager for excellent technical assistance. P.K. and S.L. were PhD students of the International Max Planck Research School Molecular and Cellular Biology and of the Faculty of Biology, University of Freiburg, M.A.V. is a PhD student of the Department of Biomedical Science, University of Sheffield. This work was supported by the Deutsche Forschungsgemeinschaft (TA–310–1, TA–310–2 and SFB592 to V.T.).

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P.K. and M.A.V. performed the experiments, analyzed the data and wrote the paper, S.L. cloned the Hes5Cre construct and performed neurosphere assays, M.M. analyzed the miRNA array data, M.M.W.C. and D.R.L. provided the floxed Drosha mice, G.M.H. and S.A.W. helped with the CLIP analysis. V.T. designed the project, performed experiments, analyzed the data and wrote the paper.

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Correspondence to Verdon Taylor.

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Knuckles, P., Vogt, M., Lugert, S. et al. Drosha regulates neurogenesis by controlling Neurogenin 2 expression independent of microRNAs. Nat Neurosci 15, 962–969 (2012). https://doi.org/10.1038/nn.3139

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