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.

Canonical microRNAs Enable Differentiation, Protect Against DNA Damage, and Promote Cholesterol Biosynthesis in Neural Stem Cells

“…S1). qRT-PCR analysis revealed that, instead of an increase of Neurog2 transcripts as reported in Drosha -/-NPCs [25], Neurog2 expression is significantly downregulated in Dgcr8 -/-NSCs (Fig. 1D).…”

“…Because it has been implicated that Drosha -/-NPCs would quickly differentiate into neurons in embryonic brain due to accumulation of Neurog2 transcripts [25], we examined whether Dgcr8 -/-NSCs are similarly defective in self-renewal by long term in vitro passaging. We found that Dgcr8 -/-NSCs can be stably cultured for at least 20 passages without noticeable changes in cell morphology or stem cell marker expression ( Supplementary Fig.…”

“…Because disruption of Dicer leads to a complete loss of several small RNA species, it remains unclear how disruption of canonical miRNAs alone affects NSCs. Furthermore, it has been implicated that Drosha -/-NPCs would quickly undergo neuronal differentiation because of accumulation of Neurog2 transcripts, which encode a differentiation-promoting transcription factor and are cleaved by the Drosha-Dgcr8 microprocessor [25]. Therefore, it is not entirely clear whether NSCs lacking microprocessor activities can self-renew and be stably maintained in culture.…”

Microprocessor Complex Subunit DiGeorge Syndrome Critical Region Gene 8 (Dgcr8) Is Required for Schwann Cell Myelination and Myelin Maintenance

“…Indeed, a well recognized role of miRNAs is to function as a fail-safe mechanism to eliminate the effect of baseline levels of transcription (81,83,84). Second, it is also possible that DGCR8 itself directly destabilizes certain mRNAs (85), and such targets would be derepressed in Dgcr8 cKO mutants. Third, denervation-associated genes may be up-regulated indirectly due to the loss of axons or the receipt of axonal signals.…”

“…In the early stages of mammalian forebrain neurogenesis, the Microprocessor complex directly binds to and destabilizes the Neurog2 mRNA. Ablating DGCR8 in this system stabilizes the expression of such proneural genes and leads to precocious differentiation that is not seen in Dicer1 knockouts (85). Indeed, DICER1-dependent and DGCR8-independent non-canonical miRNAs, such as endogenous shRNA, siRNA, mirtrons, and H/ACA small nucleolar RNA-derived small RNA, have also been shown to underpin phenotypes that are more severe in Dicer1 knockouts than in Dgcr8 knockouts (88 -90).…”