Hypoxia-induced apoptosis of astrocytes is mediated by reduction of Dicer and activation of caspase-1.

Hypoxia is a condition in which the whole body or a region of the body is deprived of oxygen supply. The brain is very sensitive to the lack of oxygen and cerebral hypoxia can rapidly cause severe brain damage. Astrocytes are essential for the survival and function of neurons. Therefore, protecting astrocytes against cell death is one of the main therapeutic strategies for treating hypoxia. Hence, the mechanism of hypoxia-induced astrocytic cell death should be fully elucidated. In this study, astrocytes were exposed to hypoxic conditions using a hypoxia work station or the hypoxia mimetic agent cobalt chloride (CoCl2 ). Both the hypoxic gas mixture (1% O2 ) and chemical hypoxia-induced apoptotic cell death in T98G glioblastoma cells and mouse primary astrocytes. Reactive oxygen species were generated in response to the hypoxia-mediated activation of caspase-1. Active caspase-1 induced the classical caspase-dependent apoptosis of astrocytes. In addition, the microRNA processing enzyme Dicer was cleaved by caspase-3 during hypoxia. Knockdown of Dicer using antisense oligonucleotides induced apoptosis of T98G cells. Taken together, these results suggest that astrocytic cell death during hypoxia is mediated by the reactive oxygen species/caspase-1/classical caspase-dependent apoptotic pathway. In addition, the decrease in Dicer levels by active caspase-3 amplifies this apoptotic pathway via a positive feedback loop. These findings may provide a new target for therapeutic interventions in cerebral hypoxia.

DROSHA-Dependent AIM2 Inflammasome Activation Contributes to Lung Inflammation during Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) has been linked to chronic lung inflammation. Drosha ribonuclease III (DROSHA), a class 2 ribonuclease III enzyme, plays a key role in microRNA (miRNA) biogenesis. However, the mechanisms by which DROSHA affects the lung inflammation during idiopathic pulmonary fibrosis (IPF) remain unclear. Here, we demonstrate that DROSHA regulates the absent in melanoma 2 (AIM2) inflammasome activation during idiopathic pulmonary fibrosis (IPF). Both DROSHA and AIM2 protein expression were elevated in alveolar macrophages of patients with IPF. We also found that DROSHA and AIM2 protein expression were increased in alveolar macrophages of lung tissues in a mouse model of bleomycin-induced pulmonary fibrosis. DROSHA deficiency suppressed AIM2 inflammasome-dependent caspase-1 activation and interleukin (IL)-1ß and IL-18 secretion in primary mouse alveolar macrophages and bone marrow-derived macrophages (BMDMs). Transduction of microRNA (miRNA) increased the formation of the adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks, which is required for AIM2 inflammasome activation in BMDMs. Our results suggest that DROSHA promotes AIM2 inflammasome activation-dependent lung inflammation during IPF.

Dicer1 facilitates liver regeneration in a manner dependent on the inhibitory effect of miR-21 on Pten and Rhob expression.

AIMS: Tamoxifen-induced liver-specific Dicer1 deletion (iDicer1-/-) in mature mice may provide clues demonstrating the genuine effects of acute loss of Dicer1 and miRNAs in the liver regeneration process. MAIN METHODS: In this study, mice with tamoxifen-induced Dicer1 deletion through the Cre/LoxP system were constructed and then underwent classic 70% partial hepatectomy or CCl4-induced liver injury. To rescue the inhibitory effect of Dicer1 ablation on liver regeneration, miR-21 agomir was injected into the tail vein of iDicer1-/- mice. KEY FINDINGS: Unlike constitutive embryonic deletion of Dicer1, tamoxifen-induced Dicer1 deletion did not result in severe liver injury or lesions, providing an ideal model for investigating acute loss of Dicer1 and miRNAs in liver regeneration. Dicer1 deletion led to impaired liver regeneration through the inhibitory effect of miR-21 on PTEN and Rhob expression. SIGNIFICANCE: In our previous study, we found that embryonic loss of Dicer1 impairs hepatocyte survival and leads to chronic inflammation and progenitor cell activation, while the role of Dicer1 in liver regeneration remains largely unknown. We clearly identified the promotion effect of Dicer1 on liver regeneration by increasing miR-21 expression, which inhibits the expression of two negative cell proliferation regulators, Pten and Rhob.

MiR-34 and MiR-200: Regulator of Cell Fate Plasticity and Neural Development.

Studies from last two decades have established microRNAs (miRNAs) as the most influential regulator of gene expression, especially at the post-transcriptional stage. The family of small RNA molecules including miRNAs is highly conserved and expressed throughout the multicellular organism. MiRNAs regulate gene expression by binding to 3' UTR of protein-coding mRNAs and initiating either decay or movement of mRNAs to stress granules. Tissues or cells, which go through cell fate transformation like stem cells, brain cells, iPSCs, or cancer cells show very dynamic expression profile of miRNAs. Inability to pass the developmental stages of Dicer (miRNA maturation enzyme) knockout animals has confirmed that expression of mature and functional miRNAs is essential for proper development of different organs and tissues. Studies from our laboratory and elsewhere have demonstrated the role of miR-200 and miR-34 families in neural development and have shown higher expression of both families in mature and differentiated neurons. In present review, we have provided a general overview of miRNAs and focused on the role of miR-34 and miR-200, two miRNA families, which have the capability to change the phenotype and fate of a cell in different tissues and situations.

RNAi drives nonreciprocal translocations at eroding chromosome ends to establish telomere-free linear chromosomes.

The identification of telomerase-negative HAATI (heterochromatin amplification-mediated and telomerase-independent) cells, in which telomeres are superseded by nontelomeric heterochromatin tracts, challenged the idea that canonical telomeres are essential for chromosome linearity and raised crucial questions as to how such tracts translocate to eroding chromosome ends and confer end protection. Here we show that HAATI arises when telomere loss triggers a newly recognized illegitimate translocation pathway that requires RNAi factors. While RNAi is necessary for the translocation events that mobilize ribosomal DNA (rDNA) tracts to all chromosome ends (forming "HAATIrDNA" chromosomes), it is dispensable for HAATIrDNA maintenance. Surprisingly, Dicer (Dcr1) plays a separate, RNAi-independent role in preventing formation of the rare HAATI subtype in which a different repetitive element (the subtelomeric element) replaces telomeres. Using genetics and fusions between shelterin components and rDNA-binding proteins, we mapped the mechanism by which rDNA loci engage crucial end protection factors-despite the absence of telomere repeats-and secure end protection. Sequence analysis of HAATIrDNA genomes allowed us to propose RNA and DNA polymerase template-switching models for the mechanism of RNAi-triggered rDNA translocations. Collectively, our results reveal unforeseen roles for noncoding RNAs (ncRNAs) in assembling a telomere-free chromosome end protection device.

MicroRNA-21 and Dicer are dispensable for hepatic stellate cell activation and the development of liver fibrosis.

Fibrosis and cancer represent two major complications of chronic liver disease. MicroRNAs have been implicated in the development of fibrosis and cancer, thus constituting potential therapeutic targets. Here, we investigated the role of microRNA-21 (miR-21), a microRNA that has been implicated in the development of fibrosis in multiple organs and has also been suggested to act as an "oncomir." Accordingly, miR-21 was the microRNA that showed the strongest up-regulation in activated hepatic stellate cells (HSCs) in multiple models of fibrogenesis, with an 8-fold to 24-fold induction compared to quiescent HSCs. However, miR-21 antisense inhibition did not suppress the activation of murine or human HSCs in culture or in liver slices. Moreover, genetic deletion of miR-21 in two independently generated knockout mice or miR-21 antisense inhibition did not alter HSC activation or liver fibrosis in models of toxic and biliary liver injury. Despite a strong up-regulation of miR-21 in injury-associated hepatocellular carcinoma and in cholangiocarcinoma, miR-21 deletion or antisense inhibition did not reduce the development of liver tumors. As inhibition of the most up-regulated microRNA did not affect HSC activation, liver fibrosis, or fibrosis-associated liver cancer, we additionally tested the role of microRNAs in HSCs by HSC-specific Dicer deletion. Although Dicer deletion decreased microRNA expression in HSCs and altered the expression of select genes, it only exerted negligible effects on HSC activation and liver fibrosis. CONCLUSION: Genetic and pharmacologic manipulation of miR-21 does not inhibit the development of liver fibrosis and liver cancer. Moreover, suppression of microRNA synthesis does not significantly affect HSC phenotype and activation. (Hepatology 2018;67:2414-2429).

The insertion in the double-stranded RNA binding domain of human Drosha is important for its function.

microRNAs (miRNAs) are first transcribed as long, primary transcripts, which are then processed by multiple enzymes and proteins to generate the single-stranded, approximately 22-nucleotide (nt)-long mature miRNAs. A critical step in animal miRNA biogenesis is the cleavage of primary miRNA transcripts (pri-miRNAs) to produce precursor miRNAs (pre-miRNAs) by the enzyme Drosha. How Drosha recognizes its substrates remains incompletely understood. In this study we constructed a series of human Drosha mutants and examined their enzymatic activities and interaction with RNAs. We found that the N-terminal region is required for the nuclear localization and cellular function of Drosha. And in contrast to previous reports, we showed that the double-stranded RNA binding domain (RBD) of Drosha exhibited a weak but noticeable affinity for RNA. Compared to the RBDs of other RNA-binding proteins, the RBD of Drosha has a short insert, whose mutations reduced RNA binding and pri-miRNA cleavage. Overexpression of Drosha RBD mutants in a reporter assay corroborated their deficiencies in Drosha activity in cell cultures. In addition, we found that point mutations in the RNaseIIIb domain of Drosha implicated in Wilms tumors differentially affected cleavage of the 5' and 3' strands of pri-miRNAs in vitro. In conclusion, our results provided important insights into the mechanism of pri-miRNA processing by human Drosha.

Dicer regulates non-homologous end joining and is associated with chemosensitivity in colon cancer patients.

DNA double-strand break (DSB) repair is an important mechanism underlying chemotherapy resistance in human cancers. Dicer participates in DSB repair by facilitating homologous recombination. However, whether Dicer is involved in non-homologous end joining (NHEJ) remains unknown. Here, we addressed whether Dicer regulates NHEJ and chemosensitivity in colon cancer cells. Using our recently developed NHEJ assay, we found that DSB introduction by I-SceI cleavage leads to Dicer upregulation. Dicer knockdown increased SIRT7 binding and decreased the level of H3K18Ac (acetylated lysine 18 of histone H3) at DSB sites, thereby repressing the recruitment of NHEJ factors to DSB sites and inhibiting NHEJ. Dicer overexpression reduced SIRT7 binding and increased the level of H3K18Ac at DSB sites, promoting the recruitment of NHEJ factors to DSBs and moderately enhancing NHEJ. Dicer knockdown and overexpression increased and decreased, respectively, the chemosensitivity of colon cancer cells. Dicer protein expression in colon cancer tissues of patients was directly correlated with chemoresistance. Our findings revealed a function of Dicer in NHEJ-mediated DSB repair and the association of Dicer expression with chemoresistance in colon cancer patients.

Dicer1 dysfunction promotes stemness and aggression in endometrial carcinoma.

Endometrial carcinoma is one of the most common gynecological malignancies, but the molecular events involved in the development and progression of endometrial carcinoma remain unclear. Dicer1 and cancer stem cells play important roles in cell motility and survival. This study investigated the role of the let-7 family and Dicer1 in the stemness of endometrial carcinoma cells. We profiled Dicer1 expression in clinical samples and explored its relationship with stem cell-associated markers and clinical parameters. We showed that Dicer1 dysfunction leads to the enrichment of tumor stemness features and tumor aggression both in vitro and in vivo. We also identified the mechanism related to this potential tumor-predisposing phenotype: loss of Dicer1 induced abnormal expression of the let-7 family, which comprises well-known tumor suppressors, thus regulating stemness in endometrial carcinoma cells.

Dicer loss and recovery induce an oncogenic switch driven by transcriptional activation of the oncofetal Imp1-3 family.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression critical for organismal viability. Changes in miRNA activity are common in cancer, but how these changes relate to subsequent alterations in transcription and the process of tumorigenesis is not well understood. Here, we report a deep transcriptional, oncogenic network regulated by miRNAs. We present analysis of the gene expression and phenotypic changes associated with global miRNA restoration in miRNA-deficient fibroblasts. This analysis uncovers a miRNA-repressed network containing oncofetal genes Imp1, Imp2, and Imp3 (Imp1-3) that is up-regulated primarily transcriptionally >100-fold upon Dicer loss and is resistant to resilencing by complete restoration of miRNA activity. This Dicer-resistant epigenetic switch confers tumorigenicity to these cells. Let-7 targets Imp1-3 are required for this tumorigenicity and feed back to reinforce and sustain expression of the oncogenic network. Together, these Dicer-resistant genes constitute an mRNA expression signature that is present in numerous human cancers and is associated with poor survival.

Epithelialization of mouse ovarian tumor cells originating in the fallopian tube stroma.

Epithelial ovarian carcinoma accounts for 90% of all ovarian cancer and is the most deadly gynecologic malignancy. Recent studies have suggested that fallopian tube fimbriae can be the origin of cells for high-grade serous subtype of epithelial ovarian carcinoma (HGSOC). A mouse HGSOC model with conditional Dicer-Pten double knockout (Dicer-Pten DKO) developed primary tumors, intriguingly, from the fallopian tube stroma. We examined the growth and epithelial phenotypes of the Dicer-Pten DKO mouse tumor cells contributable by each gene knockout. Unlike human ovarian epithelial cancer cells that expressed full-length E-cadherin, the Dicer-Pten DKO stromal tumor cells expressed cleaved E-cadherin fragments and metalloproteinase 2, a mixture of epithelial and mesenchymal markers. Although the Dicer-Pten DKO tumor cells lost the expression of mature microRNAs as expected, they showed high levels of tRNA fragment expression and enhanced AKT activation due to the loss of PTEN function. Introduction of a Dicer1-expressing construct into the DKO mouse tumor cells significantly reduced DNA synthesis and the cell growth rate, with concurrent diminished adhesion and ZO1 epithelial staining. Hence, it is likely that the loss of Dicer promoted mesenchymal-epithelial transition in fallopian tube stromal cells, and in conjunction with Pten loss, further promoted cell proliferation and epithelial-like tumorigenesis.

Artificial small RNA for sequence specific cleavage of target RNA through RNase III endonuclease Dicer.

CRISPR-Cas9 system uses a guide RNA which functions in conjunction with Cas9 proteins to target a DNA and cleaves double-strand DNA. This phenomenon raises a question whether an artificial small RNA (asRNA), composed of a Dicer-binding RNA element and an antisense RNA, could also be used to induce Dicer to process and degrade a specific RNA. If so, we could develop a new method which is named DICERi for gene silencing or RNA editing. To prove the feasibility of asRNA, we selected MALAT-1 as target and used Hela and MDA-MB-231 cells as experimental models. The results of qRT-PCR showed that the introduction of asRNA decreased the relative expression level of target gene significantly. Next, we analyzed cell proliferation using CCK-8 and EdU staining assays, and then cell migration using wound scratch and Transwell invasion assays. We found that cell proliferation and cell migration were both suppressed remarkably after asRNA was expressed in Hela and MDA-MB-231 cells. Cell apoptosis was also detected through Hoechst staining and ELISA assays and the data indicated that he numbers of apoptotic cell in experimental groups significantly increased compared with negative controls. In order to prove that the gene silencing effects were caused by Dicer, we co-transfected shRNA silencing Dicer and asRNA. The relative expression levels of Dicer and MALAT-1 were both detected and the results indicated that when the cleavage role of Dicer was silenced, the relative expression level of MALAT-1 was not affected after the introduction of asRNA. All the above results demonstrated that these devices directed by Dicer effectively excised target RNA and repressed the target genes, thus causing phenotypic changes. Our works adds a new dimension to gene regulating technologies and may have broad applications in construction of gene circuits.

MicroRNA Biogenesis and Hedgehog-Patched Signaling Cooperate to Regulate an Important Developmental Transition in Granule Cell Development.

The Dicer1, Dcr-1 homolog (Drosophila) gene encodes a type III ribonuclease required for the canonical maturation and functioning of microRNAs (miRNAs). Subsets of miRNAs are known to regulate normal cerebellar granule cell development, in addition to the growth and progression of medulloblastoma, a neoplasm that often originates from granule cell precursors. Multiple independent studies have also demonstrated that deregulation of Sonic Hedgehog (Shh)-Patched (Ptch) signaling, through miRNAs, is causative of granule cell pathologies. In the present study, we investigated the genetic interplay between miRNA biogenesis and Shh-Ptch signaling in granule cells of the cerebellum by way of the Cre/lox recombination system in genetically engineered models of Mus musculus (mouse). We demonstrate that, although the miRNA biogenesis and Shh-Ptch-signaling pathways, respectively, regulate the opposing growth processes of cerebellar hypoplasia and hyperplasia leading to medulloblastoma, their concurrent deregulation was nonadditive and did not bring the growth phenotypes toward an expected equilibrium. Instead, mice developed either hypoplasia or medulloblastoma, but of a greater severity. Furthermore, some genotypes were bistable, whereby subsets of mice developed hypoplasia or medulloblastoma. This implies that miRNAs and Shh-Ptch signaling regulate an important developmental transition in granule cells of the cerebellum. We also conclusively show that the Dicer1 gene encodes a haploinsufficient tumor suppressor gene for Ptch1-induced medulloblastoma, with the monoallielic loss of Dicer1 more severe than biallelic loss. These findings exemplify how genetic interplay between pathways may produce nonadditive effects with a substantial and unpredictable impact on biology. Furthermore, these findings suggest that the functional dosage of Dicer1 may nonadditively influence a wide range of Shh-Ptch-dependent pathologies.

[A negative correlation between Drosha expression and gastric adenocarcinoma malignancy and invasion].

OBJECTIVE: To investigate the role of Drosha expression in the progression of gastric adenocarcinoma and its impact on the invasive ability of SGC-7901 human gastric cancer cells. METHODS: Drosha expression was detected in 889 gastric carcinoma samples on tissue microarrays by immunohistochemistry staining and quantified by Image-Pro Plus software. Statistical analysis was used to evaluate the correlations between Drosha expression and the clinicopathological characteristics of the 889 tumor cases or the outcomes of 309 gastric adenocarcinoma patients. Drosha was knocked down in SGC-7901 cells by small interfering RNA (siRNA), and cell invasive ability was assessed by Transwell(TM) assay. RESULTS: Drosha expression was the highest in the well differentiated gastric adenocarcinoma (median absorbance, 0.4195), and the lowest in the poorly differentiated samples. Drosha expression was significantly related to Laren classification, tumor size, tumor invasion depth, lymph node metastasis, tumor pathological grade and stage. Patients in Drosha positive group had a higher survival rate than those in Drosha negative group. Silencing Drosha in SGC-7901 cells resulted in an enhanced cell invasion. CONCLUSION: Drosha expression was reduced gradually with the degrading histological differentiation of gastric adenocarcinoma, and the knock-down of Drosha expression could promote gastric adenocarcinoma cell invasion.

Functional and Genetic Analysis Identify a Role for Arabidopsis ARGONAUTE5 in Antiviral RNA Silencing.

RNA silencing functions as an antiviral defense through the action of DICER-like (DCL) and ARGONAUTE (AGO) proteins. In turn, plant viruses have evolved strategies to counteract this defense mechanism, including the expression of suppressors of RNA silencing. Potato virus X (PVX) does not systemically infect Arabidopsis thaliana Columbia-0, but is able to do so effectively in mutants lacking at least two of the four Arabidopsis DCL proteins. PVX can also infect Arabidopsis ago2 mutants, albeit less effectively than double DCL mutants, suggesting that additional AGO proteins may mediate anti-viral defenses. Here we show, using functional assays, that all Arabidopsis AGO proteins have the potential to target PVX lacking its viral suppressor of RNA silencing (VSR), P25, but that only AGO2 and AGO5 are able to target wild-type PVX. However, P25 directly affects only a small subset of AGO proteins, and we present evidence indicating that its protective effect is mediated by precluding AGO proteins from accessing viral RNA, as well as by directly inhibiting the RNA silencing machinery. In agreement with functional assays, we show that Potexvirus infection induces AGO5 expression and that both AGO2 and AGO5 are required for full restriction of PVX infection in systemic tissues of Arabidopsis.

Vascular Response of Ruthenium Tetraamines in Aortic Ring from Normotensive Rats / Resposta Vascular de Tetra-Aminas de Rutênio em Anéis de Aorta de Ratos Normotensos

Background: Ruthenium (Ru) tetraamines are being increasingly used as nitric oxide (NO) carriers. In this context, pharmacological studies have become highly relevant to better understand the mechanism of action involved. Objective: To evaluate the vascular response of the tetraamines trans-[RuII(NH3)4(Py)(NO)]3+, trans-[RuII(Cl)(NO) (cyclan)](PF6)2, and trans-[RuII(NH3)4(4-acPy)(NO)]3+. Methods: Aortic rings were contracted with noradrenaline (10−6 M). After voltage stabilization, a single concentration (10−6 M) of the compounds was added to the assay medium. The responses were recorded during 120 min. Vascular integrity was assessed functionally using acetylcholine at 10−6 M and sodium nitroprusside at 10−6 M as well as by histological examination. Results: Histological analysis confirmed the presence or absence of endothelial cells in those tissues. All tetraamine complexes altered the contractile response induced by norepinephrine, resulting in increased tone followed by relaxation. In rings with endothelium, the inhibition of endothelial NO caused a reduction of the contractile effect caused by pyridine NO. No significant responses were observed in rings with endothelium after treatment with cyclan NO. In contrast, in rings without endothelium, the inhibition of guanylate cyclase significantly reduced the contractile response caused by the pyridine NO and cyclan NO complexes, and both complexes caused a relaxing effect. Conclusion: The results indicate that the vascular effect of the evaluated complexes involved a decrease in the vascular tone induced by norepinephrine (10−6 M) at the end of the incubation period in aortic rings with and without endothelium, indicating the slow release of NO from these complexes and suggesting that the ligands promoted chemical stability to the molecule. Moreover, we demonstrated that the association of Ru with NO is more stable when the ligands pyridine and cyclan ...

Fundamento: As tetra-aminas de rutênio cada vez mais se destacam como carreadoras da molécula de óxido nítrico. Desse modo, estudos farmacológicos tornam-se altamente relevantes, afim de melhor compreender o mecanismo de ação envolvido. Objetivo: Avaliar a resposta vascular das tetra-aminas trans-[RuII(NH3)4(Py)(NO)]3+, trans-[RuII(Cl)(NO)(Cyclan)](PF6)2 e trans-[RuII(NH3)4(4-acPy)(NO)]3+. Métodos: Anéis de aorta foram pré-contraídos com noradrenalina (10-6M). Após estabilização da tensão, concentração única (10-6M) dos compostos foi adicionada ao banho de incubação. As respostas foram registradas ao longo de 120 minutos. A integridade vascular foi avaliada funcionalmente (acetilcolina 10-6M; nitroprussiato de sódio 10-6M) e histologicamente Resultados: A análise histológica confirmou a presença ou não de células endoteliais nos tecidos analisados. Todos os complexos alteraram a resposta contrátil induzida pela noradrenalina, resultando em aumento de tônus seguido de efeito relaxante. Em anéis com endotélio, a inibição do óxido nítrico endotelial causou redução do efeito contrátil da piridina óxido nítrico. Não foram observadas respostas significativas em anéis com endotélio referente ao composto cyclan óxido nítrico. Por outro lado, em anéis sem endotélio, a inibição da guanilato ciclase reduziu significativamente a resposta contrátil dos complexos piridina óxido nítrico e cyclan óxido nítrico, levando ambos os compostos a um efeito relaxante. Conclusão: Os resultados obtidos demonstram que o efeito vascular dos complexos avaliados apresentaram diminuição no tônus vascular induzido pela noradrenalina (10-6M) ao final do tempo de incubação, em anéis com e sem endotélio, indicando liberação lenta da molécula de óxido nítrico do composto estudado e sugerindo que os ligantes causaram estabilidade química à molécula. Demonstramos que a ligação rutênio óxido nítrico é mais estável quando utilizamos os ligantes piridina e cyclan para a formulação ...

Micro(RNA) managing by mTORC1.

In this issue of Molecular Cell, Ye et al. (2015) demonstrate that mTORC1 globally regulates miRNA biogenesis under nutrient-rich conditions via the E3 ubiquitin ligase Mdm2, which promotes Drosha degradation.

An mTORC1-Mdm2-Drosha axis for miRNA biogenesis in response to glucose- and amino acid-deprivation.

mTOR senses nutrient and energy status to regulate cell survival and metabolism in response to environmental changes. Surprisingly, targeted mutation of Tsc1, a negative regulator of mTORC1, caused a broad reduction in miRNAs due to Drosha degradation. Conversely, targeted mutation of Raptor, an essential component of mTORC1, increased miRNA biogenesis. mTOR activation increased expression of Mdm2, which is hereby identified as the necessary and sufficient ubiquitin E3 ligase for Drosha. Drosha was induced by nutrient and energy deprivation and conferred resistance to glucose deprivation. Using a high-throughput screen of a miRNA library, we identified four miRNAs that were necessary and sufficient to protect cells against glucose-deprivation-induced apoptosis. These miRNA was regulated by glucose through the mTORC1-MDM2-DROSHA axis. Taken together, our data reveal an mTOR-Mdm2-Drosha pathway in mammalian cells that broadly regulates miRNA biogenesis as a response to alteration in cellular environment.

Early postnatal ablation of the microRNA-processing enzyme, Drosha, causes chondrocyte death and impairs the structural integrity of the articular cartilage.

OBJECTIVE: In growth plate chondrocytes, loss of Dicer, a microRNA (miRNA)-processing enzyme, causes defects in proliferation and differentiation, leading to a lethal skeletal dysplasia. However roles of miRNAs in articular chondrocytes have not been defined in vivo. To investigate the role of miRNAs in articular chondrocytes and to explore the possibility of generating a novel mouse osteoarthritis (OA) model caused by intrinsic cellular dysfunction, we ablated Drosha, another essential enzyme for miRNA biogenesis, exclusively in articular chondrocytes of postnatal mice. DESIGN: First, to confirm that the essential role of miRNAs in skeletal development, we ablated the miRNA biogenesis pathway by deleting Drosha or DGCR8 in growth plate chondrocytes. Next, to investigate the role of miRNAs in articular cartilage, we deleted Drosha using Prg4-CreER(T) transgenic mice expressing a tamoxifen-activated Cre recombinase (CreER(T)) exclusively in articular chondrocytes. Tamoxifen was injected at postnatal days, 7, 14, 21, and 28 to ablate Drosha. RESULTS: Deletion of Drosha or DGCR8 in growth plate chondrocytes caused a lethal skeletal defect similar to that of Dicer deletion, confirming the essential role of miRNAs in normal skeletogenesis. Early postnatal Drosha deletion in articular chondrocytes significantly increased cell death and decreased Safranin-O staining. Mild OA-like changes, including surface erosion and cleft formation, were found in male mice at 6 months of age; however such changes in females were not observed even at 9 months of age. CONCLUSIONS: Early postnatal Drosha deficiency induces articular chondrocyte death and can cause a mild OA-like pathology.

Global microRNA expression is essential for murine mast cell development in vivo.

MicroRNAs (miRNAs) are small, noncoding RNAs that have been shown to play a critical role in normal physiology and disease, such as hematopoietic development and cancer. However, their role in mast-cell function and development is poorly understood. The major objective of this study was to determine how global miRNA expression affects mast-cell physiology. The RNase III endonuclease, Dicer, is required for the processing of pre-miRNAs into mature miRNAs. To investigate the effect of global miRNA depletion on mast cells in vivo, we generated a mast-cell-specific knock out of Dicer in mice. Transgenic mice (Mcpt5-Cre) that express Cre selectively in connective tissue mast cells were crossed with mice carrying the floxed conditional Dicer allele (Dicer fl/fl). Mcpt5-Cre × Dicer fl/fl mice with homozygous Dicer gene deletion in mast cells were found to have a profound mast-cell deficiency with near complete loss of peritoneal, gastrointestinal, and skin mast cells. We examined the in vivo functional consequence of mast-cell-specific Dicer deletion using an immunoglobulin-E-dependent passive systemic anaphylaxis murine model. Immunoglobulin-E-sensitized wild type Mcpt5-Cre × Dicer +/+ and heterozygous Mcpt5-Cre × Dicer fl/+ mice show marked hypothermia with antigen; however, homozygous Mcpt5-Cre × Dicer fl/fl mice were completely unresponsive to antigen challenge. These studies suggest a critical role for Dicer and miRNA expression for establishment of tissue compartments of functional mast cells in vivo.