Essential role of the amino-terminal region of Drosha for the Microprocessor function.

Drosha is a core component of the Microprocessor complex that cleaves primary-microRNAs (pri-miRNAs) to generate precursor-miRNA and regulates the expression of ∼80 ribosomal protein (RP) genes. Despite the fact that mutations in the amino-terminal region of Drosha (Drosha-NTR) are associated with a vascular disorder, hereditary hemorrhagic telangiectasia, the precise function of Drosha-NTR remains unclear. By deleting exon 5 from the Drosha gene and generating a Drosha mutant lacking the NTR (ΔN), we demonstrate that ΔN is unable to process pri-miRNAs, which leads to a global miRNA depletion, except for the miR-183/96/182 cluster. We find that Argonaute 2 facilitates the processing of the pri-miR-183/96/182 in ΔN cells. Unlike full-length Drosha, ΔN is not degraded under serum starvation, resulting in unregulated RP biogenesis and protein synthesis in ΔN cells, allowing them to evade growth arrest. This study reveals the essential role of Drosha-NTR in miRNA production and nutrient-dependent translational control.

Reversal of pulmonary veno-occlusive disease phenotypes by inhibition of the integrated stress response.

Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary hypertension arising from EIF2AK4 gene mutations or mitomycin C (MMC) administration. The lack of effective PVOD therapies is compounded by a limited understanding of the mechanisms driving the vascular remodeling in PVOD. We show that the administration of MMC in rats mediates the activation of protein kinase R (PKR) and the integrated stress response (ISR), which lead to the release of the endothelial adhesion molecule VE-Cadherin in the complex with Rad51 to the circulation, disruption of endothelial barrier, and vascular remodeling. Pharmacological inhibition of PKR or ISR attenuates the depletion of VE-Cadherin, elevation of vascular permeability, and vascular remodeling instigated by MMC, suggesting potential clinical intervention for PVOD. Finally, the severity of PVOD phenotypes was increased by a heterozygous BMPR2 mutation that truncates the carboxyl tail of BMPR2, underscoring the role of deregulated BMP signal in the development of PVOD.

Structural basis for ALK2/BMPR2 receptor complex signaling through kinase domain oligomerization.

Upon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs.

Control of ribosomal protein synthesis by the Microprocessor complex.

Ribosome biogenesis in eukaryotes requires the coordinated production and assembly of 80 ribosomal proteins and four ribosomal RNAs (rRNAs), and its rate must be synchronized with cellular growth. Here, we showed that the Microprocessor complex, which mediates the first step of microRNA processing, potentiated the transcription of ribosomal protein genes by eliminating DNA/RNA hybrids known as R-loops. Nutrient deprivation triggered the nuclear export of Drosha, a key component of the Microprocessor complex, and its subsequent degradation by the E3 ubiquitin ligase Nedd4, thereby reducing ribosomal protein production and protein synthesis. In mouse erythroid progenitors, conditional deletion of Drosha led to the reduced production of ribosomal proteins, translational inhibition of the mRNA encoding the erythroid transcription factor Gata1, and impaired erythropoiesis. This phenotype mirrored the clinical presentation of human "ribosomopathies." Thus, the Microprocessor complex plays a pivotal role in synchronizing protein synthesis capacity with cellular growth rate and is a potential drug target for anemias caused by ribosomal insufficiency.

Bone morphogenetic protein signaling is required for RAD51-mediated maintenance of genome integrity in vascular endothelial cells.

The integrity of blood vessels is fundamental to vascular homeostasis. Inactivating mutations in the bone morphogenetic protein (BMP) receptor type II (BMPR2) gene cause hereditary vascular disorders, including pulmonary arterial hypertension and hereditary hemorrhagic telangiectasia, suggesting that BMPR2 and its downstream signaling pathway are pivotal to the maintenance of vascular integrity through an unknown molecular mechanism. Here we report that inactivation of BMPR2 in pulmonary vascular endothelial cells results in a deficit of RAD51, an enzyme essential for DNA repair and replication. Loss of RAD51, which causes DNA damage and cell death, is also detected in animal models and human patients with pulmonary arterial hypertension. Restoration of BMPR2 or activation of the BMP signaling pathway rescues RAD51 and prevents DNA damage. This is an unexpected role of BMP signaling in preventing the accumulation of DNA damage and the concomitant loss of endothelial integrity and vascular remodeling associated with vascular disorders.

Inactivating mutations in Drosha mediate vascular abnormalities similar to hereditary hemorrhagic telangiectasia.

The transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) family of cytokines critically regulates vascular morphogenesis and homeostasis. Impairment of TGF-ß or BMP signaling leads to heritable vascular disorders, including hereditary hemorrhagic telangiectasia (HHT). Drosha, a key enzyme for microRNA (miRNA) biogenesis, also regulates the TGF-ß and BMP pathway through interaction with Smads and their joint control of gene expression through miRNAs. We report that mice lacking Drosha in the vascular endothelium developed a vascular phenotype resembling HHT that included dilated and disorganized vasculature, arteriovenous fistulae, and hemorrhages. Exome sequencing of HHT patients who lacked known pathogenic mutations revealed an overrepresentation of rare nonsynonymous variants of DROSHA Two of these DROSHA variants (P100L and R279L) did not interact with Smads and were partially catalytically active. In zebrafish, expression of these mutants or morpholino-directed knockdown of Drosha resulted in angiogenesis defects and abnormal vascular permeability. Together, our studies point to an essential role of Drosha in vascular development and the maintenance of vascular integrity, and reveal a previously unappreciated link between Drosha dysfunction and HHT.

Hyperactive locomotion in a Drosophila model is a functional readout for the synaptic abnormalities underlying fragile X syndrome.

Fragile X syndrome (FXS) is the most common cause of heritable intellectual disability and autism and affects ~1 in 4000 males and 1 in 8000 females. The discovery of effective treatments for FXS has been hampered by the lack of effective animal models and phenotypic readouts for drug screening. FXS ensues from the epigenetic silencing or loss-of-function mutation of the fragile X mental retardation 1 (FMR1) gene, which encodes an RNA binding protein that associates with and represses the translation of target mRNAs. We previously found that the activation of LIM kinase 1 (LIMK1) downstream of augmented synthesis of bone morphogenetic protein (BMP) type 2 receptor (BMPR2) promotes aberrant synaptic development in mouse and Drosophila models of FXS and that these molecular and cellular markers were correlated in patients with FXS. We report that larval locomotion is augmented in a Drosophila FXS model. Genetic or pharmacological intervention on the BMPR2-LIMK pathway ameliorated the synaptic abnormality and locomotion phenotypes of FXS larvae, as well as hyperactivity in an FXS mouse model. Our study demonstrates that (i) the BMPR2-LIMK pathway is a promising therapeutic target for FXS and (ii) the locomotion phenotype of FXS larvae is a quantitative functional readout for the neuromorphological phenotype associated with FXS and is amenable to the screening novel FXS therapeutics.

Let-7 microRNA-dependent control of leukotriene signaling regulates the transition of hematopoietic niche in mice.

Hematopoietic stem and progenitor cells arise from the vascular endothelium of the dorsal aorta and subsequently switch niche to the fetal liver through unknown mechanisms. Here we report that vascular endothelium-specific deletion of mouse Drosha (Drosha cKO), an enzyme essential for microRNA biogenesis, leads to anemia and death. A similar number of hematopoietic stem and progenitor cells emerge from Drosha-deficient and control vascular endothelium, but Drosha cKO-derived hematopoietic stem and progenitor cells accumulate in the dorsal aorta and fail to colonize the fetal liver. Depletion of the let-7 family of microRNAs is a primary cause of this defect, as it leads to activation of leukotriene B4 signaling and induction of the α4ß1 integrin cell adhesion complex in hematopoietic stem and progenitor cells. Inhibition of leukotriene B4 or integrin rescues maturation and migration of Drosha cKO hematopoietic stem and progenitor cells to the fetal liver, while it hampers hematopoiesis in wild-type animals. Our study uncovers a previously undefined role of innate leukotriene B4 signaling as a gatekeeper of the hematopoietic niche transition.Hematopoietic stem and progenitor cells are generated first from the vascular endothelium of the dorsal aorta and then the fetal liver but what regulates this switch is unknown. Here, the authors show that changing miRNA biogenesis and leukotriene B4 signaling in mice modulates this switch in the niche.