Cytoplasmic Drosha activity generated by alternative splicing

RNase III enzyme Drosha interacts with DGCR8 to form the Microprocessor, initiating canonical microRNA (miRNA) maturation in the nucleus. Here, we re-evaluated where Drosha functions in cells using Drosha and/or DGCR8 knock out (KO) cells and cleavage reporters. Interestingly, a truncated Drosha mutant located exclusively in the cytoplasm cleaved pri-miRNA effectively in a DGCR8-dependent manner. In addition, we demonstrated that in vitro generated pri-miRNAs when transfected into cells could be processed to mature miRNAs in the cytoplasm. These results indicate the existence of cytoplasmic Drosha (c-Drosha) activity. Although a subset of endogenous pri-miRNAs become enriched in the cytoplasm of Drosha KO cells, it remains unclear whether pri-miRNA processing is the main function of c-Drosha. We identified two novel in-frame Drosha isoforms generated by alternative splicing in both HEK293T and HeLa cells. One isoform loses the putative nuclear localization signal, generating c-Drosha. Further analysis indicated that the c-Drosha isoform is abundant in multiple cell lines, dramatically variable among different human tissues and upregulated in multiple tumors, suggesting that c-Drosha plays a unique role in gene regulation. Our results reveal a new layer of regulation on the miRNA pathway and provide novel insights into the ever-evolving functions of Drosha.     

SUMOylation at K707 of DGCR8 controls direct function of primary microRNA

DGCR8 (DiGeorge syndrome critical region gene 8) is essential for primary microRNA (pri-miRNA) processing in the cell nucleus. It specifically combines with Drosha, a nuclear RNase III enzyme, to form the Microprocessor complex (MC) that cleaves pri-miRNA to precursor miRNA (pre-miRNA), which is further processed to mature miRNA by Dicer, a cytoplasmic RNase III enzyme. Increasing evidences suggest that pri-/pre-miRNAs have direct functions in regulation of gene expression, however the underlying mechanism how it is fine-tuned remains unclear. Here we find that DGCR8 is modified by SUMO1 at the major site K⁷⁰⁷, which can be promoted by its ERK-activated phosphorylation. SUMOylation of DGCR8 enhances the protein stability by preventing the degradation via the ubiquitin proteasome pathway. More importantly, SUMOylation of DGCR8 does not alter its association with Drosha, the MC activity and miRNA biogenesis, but rather influences its affinity with pri-miRNAs. This altered affinity of DGCR8 with pri-miRNAs seems to control the direct functions of pri-miRNAs in recognition and repression of the target mRNAs, which is evidently linked to the DGCR8 function in regulation of tumorigenesis and cell migration. Collectively, our data suggest a novel mechanism that SUMOylation of DGCR8 controls direct functions of pri-miRNAs in gene silencing.     

A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting

During microRNA (miRNA) biogenesis, the Microprocessor complex (MC), composed minimally of Drosha, an RNaseIII enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary-miRNA (pri-miRNA) to release the pre-miRNA stem–loop structure. Size-exclusion chromatography of the MC, isolated from mammalian cells, suggested multiple copies of one or both proteins in the complex. However, the exact stoichiometry was unknown. Initial experiments suggested that DGCR8 bound pri-miRNA substrates specifically, and given that Drosha could not be bound or cross-linked to RNA, a sequential model for binding was established in which DGCR8 bound first and recruited Drosha. Therefore, many laboratories have studied DGCR8 binding to RNA in the absence of Drosha and have shown that deletion constructs of DGCR8 can multimerize in the presence of RNA. More recently, it was demonstrated that Drosha can bind pri-miRNA substrates in the absence of DGCR8, casting doubt on the sequential model of binding. In the same study, using a single-molecule photobleaching assay, fluorescent protein-tagged deletion constructs of DGCR8 and Drosha assembled into a heterotrimeric complex on RNA, comprising two DGCR8 molecules and one Drosha molecule. To determine the stoichiometry of Drosha and DGCR8 within the MC in the absence of added RNA, we also used a single-molecule photobleaching assay and confirmed the heterotrimeric model of the human MC. We demonstrate that a heterotrimeric complex is likely preformed in the absence of RNA and exists even when full-length proteins are expressed and purified from human cells, and when hAGT-derived tags are used rather than fluorescent proteins.     

miRNA Biogenesis Enzyme Drosha Is Required for Vascular Smooth Muscle Cell Survival

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.     

Dynamic origins of differential RNA binding function in two dsRBDs from the miRNA "microprocessor" complex

MicroRNAs (miRNAs) affect gene regulation by base pairing with mRNA and contribute to the control of cellular homeostasis. The first step in miRNA maturation is conducted in the nucleus by the "microprocessor" complex made up of an RNase III enzyme, Drosha, that contains one dsRNA binding domain (dsRBD), and DGCR8, that contains two dsRBDs in tandem. The crystal structure of DGCR8-Core (493-720), containing both dsRBDs, and the NMR solution structure of Drosha-dsRBD (1259-1337) have been reported, but the solution dynamics have not been explored for any of these dsRBDs. To better define the mechanism of dsRNA binding and thus the nuclear maturation step of miRNA processing, we report NMR spin relaxation and MD simulations of Drosha-dsRBD (1259-1337) and DGCR8-dsRBD1 (505-583). The study was motivated by electrophoretic mobility shift assays (EMSAs) of the two dsRBDs, which showed that Drosha-dsRBD does not bind a representative miRNA but isolated DGCR8-dsRBD1 does (K(d) = 9.4 ± 0.4 μM). Our results show that loop 2 in both dsRBDs is highly dynamic but the pattern of the correlations observed in MD is different for the two proteins. Additionally, the extended loop 1 of Drosha-dsRBD is more flexible than the corresponding loop in DGCR8-dsRBD1 but shows no correlation with loop 2, which potentially explains the lack of dsRNA binding by Drosha-dsRBD in the absence of the RNase III domains. The results presented in this study provide key structural and dynamic features of dsRBDs that contribute to the binding mechanism of these domains to dsRNA.     

Formation of GW bodies is a consequence of microRNA genesis

GW bodies (GWBs), or mammalian P bodies, proposed to be involved in messenger RNA storage and/or degradation, have recently been linked to RNA interference and microRNA (miRNA) processing. We report that endogenous let-7 miRNA co-precipitates with the GW182 protein complex. In addition, knockdown of two proteins, Drosha and its protein partner DGCR8, which are vital to the generation of mature miRNA, results in the loss of GWBs. Subsequent introduction of short interference RNA specific to lamin A/C is accompanied by reassembly of GWBs and concurrent knockdown of lamin A/C protein. Taken together, these studies show that miRNAs are crucial components in GWB formation.     

SNPs in human miRNA genes affect biogenesis and function

MicroRNAs (miRNAs) are 21–25-nucleotide-long, noncoding RNAs that are involved in translational regulation. Most miRNAs derive from a two-step sequential processing: the generation of pre-miRNA from pri-miRNA by the Drosha/DGCR8 complex in the nucleus, and the generation of mature miRNAs from pre-miRNAs by the Dicer/TRBP complex in the cytoplasm. Sequence variation around the processing sites, and sequence variations in the mature miRNA, especially the seed sequence, may have profound affects on miRNA biogenesis and function. In the context of analyzing the roles of miRNAs in Schizophrenia and Autism, we defined at least 24 human X-linked miRNA variants. Functional assays were developed and performed on these variants. In this study we investigate the affects of single nucleotide polymorphisms (SNPs) on the generation of mature miRNAs and their function, and report that naturally occurring SNPs can impair or enhance miRNA processing as well as alter the sites of processing. Since miRNAs are small functional units, single base changes in both the precursor elements as well as the mature miRNA sequence may drive the evolution of new microRNAs by altering their biological function. Finally, the miRNAs examined in this study are X-linked, suggesting that the mutant alleles could be determinants in the etiology of diseases.     

Crystal structure of human DGCR8 core

A complex of Drosha with DGCR8 (or its homolog Pasha) cleaves primary microRNA (pri-miRNA) substrates into precursor miRNA and initiates the microRNA maturation process. Drosha provides the catalytic site for this cleavage, whereas DGCR8 or Pasha provides a frame for anchoring substrate pri-miRNAs. To clarify the molecular basis underlying recognition of pri-miRNA by DGCR8 and Pasha, we determined the crystal structure of the human DGCR8 core (DGCR8S, residues 493-720). In the structure, the two double-stranded RNA-binding domains (dsRBDs) are arranged with pseudo two-fold symmetry and are tightly packed against the C-terminal helix. The H2 helix in each dsRBD is important for recognition of pri-miRNA substrates. This structure, together with fluorescent resonance energy transfer and mutational analyses, suggests that the DGCR8 core recognizes pri-miRNA in two possible orientations. We propose a model for DGCR8's recognition of pri-miRNA.     

A central role for the primary microRNA stem in guiding the position and efficiency of Drosha processing of a viral pri-miRNA

Processing of primary microRNA (pri-miRNA) stem–loops by the Drosha–DGCR8 complex is the initial step in miRNA maturation and crucial for miRNA function. Nonetheless, the underlying mechanism that determines the Drosha cleavage site of pri-miRNAs has remained unclear. Two prevalent but seemingly conflicting models propose that Drosha–DGCR8 anchors to and directs cleavage a fixed distance from either the basal single-stranded (ssRNA) or the terminal loop. However, recent studies suggest that the basal ssRNA and/or the terminal loop may influence the Drosha cleavage site dependent upon the sequence/structure of individual pri-miRNAs. Here, using a panel of closely related pri-miRNA variants, we further examine the role of pri-miRNA structures on Drosha cleavage site selection in cells. Our data reveal that both the basal ssRNA and terminal loop influence the Drosha cleavage site within three pri-miRNAs, the Simian Virus 40 (SV40) pri-miRNA, pri-miR-30a, and pri-miR-16. In addition to the flanking ssRNA regions, we show that an internal loop within the SV40 pri-miRNA stem strongly influences Drosha cleavage position and efficiency. We further demonstrate that the positions of the internal loop, basal ssRNA, and the terminal loop of the SV40 pri-miRNA cooperatively coordinate Drosha cleavage position and efficiency. Based on these observations, we propose that the pri-miRNA stem, defined by internal and flanking structural elements, guides the binding position of Drosha–DGCR8, which consequently determines the cleavage site. This study provides mechanistic insight into pri-miRNA processing in cells that has numerous biological implications and will assist in refining Drosha-dependent shRNA design.     

DGCR8‐mediated disruption of miRNA biogenesis induces cellular senescence in primary fibroblasts

The regulation of gene expression by microRNAs (miRNAs) is critical for normal development and physiology. Conversely, miRNA function is frequently impaired in cancer, and other pathologies, either by aberrant expression of individual miRNAs or dysregulation of miRNA synthesis. Here, we have investigated the impact of global disruption of miRNA biogenesis in primary fibroblasts of human or murine origin, through the knockdown of DGCR8, an essential mediator of the synthesis of canonical miRNAs. We find that the inactivation of DGCR8 in these cells results in a dramatic antiproliferative response, with the acquisition of a senescent phenotype. Senescence triggered by DGCR8 loss is accompanied by the upregulation of the cell‐cycle inhibitor p21CIP1. We further show that a subset of senescence‐associated miRNAs with the potential to target p21CIP1 is downregulated during DGCR8‐mediated senescence. Interestingly, the antiproliferative response to miRNA biogenesis disruption is retained in human tumor cells, irrespective of p53 status. In summary, our results show that defective synthesis of canonical microRNAs results in cell‐cycle arrest and cellular senescence in primary fibroblasts mediated by specific miRNAs, and thus identify global miRNA disruption as a novel senescence trigger.