Abstract
miRNA biogenesis enzyme Drosha cleaves double-stranded primary miRNA by interacting with double-stranded RNA binding protein DGCR8 and processes primary miRNA into precursor miRNA to participate in the miRNA biogenesis pathway. The role of Drosha in vascular smooth muscle cells (VSMCs) has not been well addressed. We generated Drosha conditional knockout (cKO) mice by crossing VSMC-specific Cre mice, SM22-Cre, with Drosha loxp/loxp mice. Disruption of Drosha in VSMCs resulted in embryonic lethality at E14.5 with severe liver hemorrhage in mutant embryos. No obvious developmental delay was observed in Drosha cKO embryos. The vascular structure was absent in the yolk sac of Drosha homozygotes at E14.5. Loss of Drosha reduced VSMC proliferation in vitro and in vivo. The VSMC differentiation marker genes, including αSMA, SM22, and CNN1, and endothelial cell marker CD31 were significantly downregulated in Drosha cKO mice compared to controls. ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated in VSMCs in vitro and in vivo. Disruption of Drosha in VSMCs of mice leads to the dysregulation of miRNA expression. Using bioinformatics approach, the interactions between dysregulated miRNAs and their target genes were analyzed. Our data demonstrated that Drosha is required for VSMC survival by targeting multiple signaling pathways.
Highlights
IntroductionDrosha is an RNAase III enzyme that interacts with the RNA binding protein DGCR8 and forms a microcomplex in the nucleus to process primary miRNAs (pri-miRNAs) into precursor miRNAs (pre-miRNAs), which are subsequently cleaved by Dicer in the cytoplasm to produce mature miRNA
Drosha is an RNAase III enzyme that interacts with the RNA binding protein DGCR8 and forms a microcomplex in the nucleus to process primary miRNAs into precursor miRNAs, which are subsequently cleaved by Dicer in the cytoplasm to produce mature miRNA
Loss of Drosha in vascular smooth muscle cells (VSMCs) resulted in multiple embryonic defects including severe liver hemorrhage, vascular wall hypoplasia, and embryonic lethality
Summary
Drosha is an RNAase III enzyme that interacts with the RNA binding protein DGCR8 and forms a microcomplex in the nucleus to process primary miRNAs (pri-miRNAs) into precursor miRNAs (pre-miRNAs), which are subsequently cleaved by Dicer in the cytoplasm to produce mature miRNA. Our studies demonstrated that DGCR8 plays a more important role than does Dicer in maintaining the VSMC functions by participating in the miRNA process in the upstream biogenesis pathway. Drosha and Dicer are two RNAase III enzymes and share the structural and functional similarity that both cleave the double-stranded RNA and have two RNAase III domains, a/b and an RNA binding domain. DGCR8 is an RNA binding protein whose structure differs from Dicer and Drosha, which do not have RNAase III domains but have two RNA binding domains. DGCR8, and Dicer are key components of the miRNA biogenesis pathway and contribute to miRNA maturation by participating in this pathway
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