Dicer In Mice Research Articles

Enhanced RNAi does not provide efficient innate antiviral immunity in mice.

In RNA interference (RNAi), long double-stranded RNA is cleaved by the Dicer endonuclease into small interfering RNAs (siRNAs), which guide degradation of complementary RNAs. While RNAi mediates antiviral innate immunity in plants and many invertebrates, vertebrates have adopted a sequence-independent response and their Dicer produces siRNAs inefficiently because it is adapted to process small hairpin microRNA precursors in the gene-regulating microRNA pathway. Mammalian endogenous RNAi is thus a rudimentary pathway of unclear significance. To investigate its antiviral potential, we modified the mouse Dicer locus to express a truncated variant (DicerΔHEL1) known to stimulate RNAi and we analyzed how DicerΔHEL1/wt mice respond to four RNA viruses: coxsackievirus B3 and encephalomyocarditis virus from Picornaviridae; tick-borne encephalitis virus from Flaviviridae; and lymphocytic choriomeningitis virus (LCMV) from Arenaviridae. Increased Dicer activity in DicerΔHEL1/wt mice did not elicit any antiviral effect, supporting an insignificant antiviral function of endogenous mammalian RNAi in vivo. However, we also observed that sufficiently high expression of DicerΔHEL1 suppressed LCMV in embryonic stem cells and in a transgenic mouse model. Altogether, mice with increased Dicer activity offer a new benchmark for identifying and studying viruses susceptible to mammalian RNAi in vivo.

Rapid downregulation of DICER is a hallmark of adipose tissue upon high-fat diet feeding

Adipose tissue regulates whole-body energy balance and is crucial for metabolic health. With energy surplus, adipose tissue expands, which may lead to local areas of hypoxia and inflammation, and consequently impair whole-body insulin sensitivity. We report that DICER, a key enzyme for miRNA maturation, is significantly lower in abdominal subcutaneous white adipose tissue of men with obesity compared with men with a lean phenotype. Furthermore, DICER is profoundly downregulated in mouse adipose tissue and liver within the first week on a high-fat diet (HFD), and remains low after prolonged HFD feeding. Downregulation of DICER in mice occurs in both mature adipocytes and stromal vascular cells. Mechanistically, chemically induced hypoxia in vitro shows DICER degradation via interaction with hypoxia-inducible factor 1-α (HIF1α). Moreover, DICER and HIF1α interact in brown adipose tissue post-HFD which may signal for DICER degradation. Finally, RNA sequencing reveals a striking time-dependent downregulation of total miRNA content in mouse subcutaneous adipose tissue after HFD feeding. Collectively, HFD in mice reduces adipose tissue DICER, likely due to hypoxia-induced interaction with HIF1α during tissue expansion, and this significantly impacts miRNA content.

Dicer, a new regulator of pluripotency exit and LINE-1 elements in mouse embryonic stem cells.

A gene regulation network orchestrates processes ensuring the maintenance of cellular identity and genome integrity. Small RNAs generated by the RNAse III DICER have emerged as central players in this network. Moreover, deletion of Dicer in mice leads to early embryonic lethality. To better understand the underlying mechanisms leading to this phenotype, we generated Dicer‐deficient mouse embryonic stem cells (mESCs). Their detailed characterization revealed an impaired differentiation potential, and incapacity to exit from the pluripotency state. We also observed a strong accumulation of LINE‐1 (L1s) transcripts, which was translated at protein level and led to an increased L1s retrotransposition. Our findings reveal Dicer as a new essential player that sustains mESCs self‐renewal and genome integrity by controlling L1s regulation.

Dicer maintains the identity and function of proprioceptive sensory neurons.

Neuronal cell identity is established during development and must be maintained throughout an animal's life (Fishell G, Heintz N. Neuron 80: 602-612, 2013). Transcription factors critical for establishing neuronal identity can be required for maintaining it (Deneris ES, Hobert O. Nat Neurosci 17: 899-907, 2014). Posttranscriptional regulation also plays an important role in neuronal differentiation (Bian S, Sun T. Mol Neurobiol 44: 359-373, 2011), but its role in maintaining cell identity is less established. To better understand how posttranscriptional regulation might contribute to cell identity, we examined the proprioceptive neurons in the dorsal root ganglion (DRG), a highly specialized sensory neuron class, with well-established properties that distinguish them from other neurons in the ganglion. By conditionally ablating Dicer in mice, using parvalbumin (Pvalb)-driven Cre recombinase, we impaired posttranscriptional regulation in the proprioceptive sensory neuron population. Knockout (KO) animals display a progressive form of ataxia at the beginning of the fourth postnatal week that is accompanied by a cell death within the DRG. Before cell loss, expression profiling shows a reduction of proprioceptor specific genes and an increased expression of nonproprioceptive genes normally enriched in other ganglion neurons. Furthermore, although central connections of these neurons are intact, the peripheral connections to the muscle are functionally impaired. Posttranscriptional regulation is therefore necessary to retain the transcriptional identity and support functional specialization of the proprioceptive sensory neurons.NEW & NOTEWORTHY We have demonstrated that selectively impairing Dicer in parvalbumin-positive neurons, which include the proprioceptors, triggers behavioral changes, a lack of muscle connectivity, and a loss of transcriptional identity as observed through RNA sequencing. These results suggest that Dicer and, most likely by extension, microRNAs are crucially important for maintaining proprioception. Additionally, this study hints at the larger question of how neurons maintain their functional and molecular specificity.

Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein.

Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse.

Dgcr8 and Dicer are essential for sex chromosome integrity during meiosis in males

Small RNAs play crucial roles in regulating gene expression during mammalian meiosis. To investigate the function of microRNAs (miRNAs) and small interfering RNAs (siRNAs) during meiosis in males, we generated germ-cell-specific conditional deletions of Dgcr8 and Dicer in mice. Analysis of spermatocytes from both conditional knockout lines revealed that there were frequent chromosomal fusions during meiosis, always involving one or both sex chromosomes. RNA sequencing indicates upregulation of Atm in spermatocytes from miRNA-deficient mice, and immunofluorescence imaging demonstrates an increased abundance of activated ATM kinase and mislocalization of phosphorylated MDC1, an ATM phosphorylation substrate. The Atm 3′UTR contains many potential microRNA target sites, and, notably, target sites for several miRNAs depleted in both conditional knockout mice were highly effective at promoting repression. RNF8, a telomere-associated protein whose localization is controlled by the MDC1–ATM kinase cascade, normally associates with the sex chromosomes during pachytene, but in both conditional knockouts redistributed to the autosomes. Taken together, these results suggest that Atm dysregulation in microRNA-deficient germ lines contributes to the redistribution of proteins involved in chromosomal stability from the sex chromosomes to the autosomes, resulting in sex chromosome fusions during meiotic prophase I.

NF-κB-DICER-miRs Axis Regulates TNF-α Expression in Responses to Endotoxin Stress

Unbalanced tumor necrosis factor (TNF)-α production is associated with pathogenesis of a variety of human diseases. However, the molecular pathways maintaining TNF-α homeostasis remain elusive. Here, we report that NF-κB/p65-DICER-miRs axis negatively regulates TNF-α production. We demonstrated that NF-κB bound to DICER promoter and transcriptionally regulated DICER expression. In addition, the NF-κB/DICER signaling suppresses TNF-α expression by generating mature forms of miR-125b and miR-130a which negatively regulate TNF-α mRNA. Furthermore, we showed that the hepatocyte-specific depletion of Dicer in mice resulted in TNF-α overproduction and sensitized the mice to endotoxin, which could be corrected by administration of miR-125b mimics. These data suggest that NF-κB/p65-DICER-miRs axis involved in maintaining of TNF-α homeostasis, and injection of miR-125b as a potential therapeutic method for septic shock.

Dgcr8 and Dicer are essential for sex chromosome integrity during meiosis in males.

ABSTRACTSmall RNAs play crucial roles in regulating gene expression during mammalian meiosis. To investigate the function of microRNAs (miRNAs) and small interfering RNAs (siRNAs) during meiosis in males, we generated germ-cell-specific conditional deletions of Dgcr8 and Dicer in mice. Analysis of spermatocytes from both conditional knockout lines revealed that there were frequent chromosomal fusions during meiosis, always involving one or both sex chromosomes. RNA sequencing indicates upregulation of Atm in spermatocytes from miRNA-deficient mice, and immunofluorescence imaging demonstrates an increased abundance of activated ATM kinase and mislocalization of phosphorylated MDC1, an ATM phosphorylation substrate. The Atm 3′UTR contains many potential microRNA target sites, and, notably, target sites for several miRNAs depleted in both conditional knockout mice were highly effective at promoting repression. RNF8, a telomere-associated protein whose localization is controlled by the MDC1–ATM kinase cascade, normally associates with the sex chromosomes during pachytene, but in both conditional knockouts redistributed to the autosomes. Taken together, these results suggest that Atm dysregulation in microRNA-deficient germ lines contributes to the redistribution of proteins involved in chromosomal stability from the sex chromosomes to the autosomes, resulting in sex chromosome fusions during meiotic prophase I.

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 invivo, 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 invivo 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 invivo.

Mir-29 Repression in Bladder Outlet Obstruction Contributes to Matrix Remodeling and Altered Stiffness

Recent work has uncovered a role of the microRNA (miRNA) miR-29 in remodeling of the extracellular matrix. Partial bladder outlet obstruction is a prevalent condition in older men with prostate enlargement that leads to matrix synthesis in the lower urinary tract and increases bladder stiffness. Here we tested the hypothesis that miR-29 is repressed in the bladder in outlet obstruction and that this has an impact on protein synthesis and matrix remodeling leading to increased bladder stiffness. c-Myc, NF-κB and SMAD3, all of which repress miR-29, were activated in the rat detrusor following partial bladder outlet obstruction but at different times. c-Myc and NF-κB activation occurred early after obstruction, and SMAD3 phosphorylation increased later, with a significant elevation at 6 weeks. c-Myc, NF-κB and SMAD3 activation, respectively, correlated with repression of miR-29b and miR-29c at 10 days of obstruction and with repression of miR-29c at 6 weeks. An mRNA microarray analysis showed that the reduction of miR-29 following outlet obstruction was associated with increased levels of miR-29 target mRNAs, including mRNAs for tropoelastin, the matricellular protein Sparc and collagen IV. Outlet obstruction increased protein levels of eight out of eight examined miR-29 targets, including tropoelastin and Sparc. Transfection of human bladder smooth muscle cells with antimiR-29c and miR-29c mimic caused reciprocal changes in target protein levels in vitro. Tamoxifen inducible and smooth muscle-specific deletion of Dicer in mice reduced miR-29 expression and increased tropoelastin and the thickness of the basal lamina surrounding smooth muscle cells in the bladder. It also increased detrusor stiffness independent of outlet obstruction. Taken together, our study supports a model where the combined repressive influences of c-Myc, NF-κB and SMAD3 reduce miR-29 in bladder outlet obstruction, and where the resulting drop in miR-29 contributes to matrix remodeling and altered passive mechanical properties of the detrusor.

Global microRNA expression is essential for mast cell development in vivo (177.18)

Abstract microRNAs (miRNAs) are small, non-coding RNAs that have been shown to play a critical role in both normal physiology and disease, such as hematopoietic development and cancer. However, their role in mast cell function and development is unexplored. The RNaseIII endonuclease, Dicer, is required for the processing of pre-miRNAs into mature miRNAs. To investigate the role 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 mast cells were crossed with mice containing the floxed conditional Dicer allele (Dicer f/f). Mice with both homozygous (Dicer -/-) and heterozygous (Dicer f/-) mast cell-specific deletion of Dicer were viable and healthy. However, Dicer -/- mice 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 IgE-dependent passive systemic anaphylaxis (PSA) murine model. IgE sensitized wild type (Dicer f/f) and heterozygous (Dicer f/-) mice show marked hypothermia with antigen; however, homozygous (Dicer -/-) mice were completely unreactive to antigen challenge. These studies suggest a critical role for Dicer and miRNA expression for establishment of mast cell tissue compartments in vivo.

MiR-143 and miR-145

Vascular smooth muscle cells (VSMCs) are able to perform both contractile and synthetic functions, which are associated with changes in morphology, proliferation, and migration rates and are characterized by the specific expression of different marker proteins. Under normal physiological conditions, VSMC rarely proliferate in adult tissues, but undergo major phenotypic changes from the contractile to the synthetic in response to environmental cues, a phenomenon known as switching, or phenotypic modulation.1,2 Phenotypic switching is accompanied by production of abundant cytokines, extracellular matrix, and an increased rate of proliferation and migration. Therefore, the transition of VSMCs from a differentiated phenotype to a dedifferentiated state plays a critical role in the pathogenesis of cardiovascular diseases such as hypertension, vascular injury, and arteriosclerosis.2,3 However, the molecular mechanisms involved in phenotypic switching remain elusive. The last decade has witnessed an exciting discovery that led to a revolution in our understanding of the extensive regulatory gene expression networks modulated by small, untranslated RNAs, microRNAs (miRNAs).4 miRNAs comprise a novel class of endogenous, small RNAs of ≈20 to 25 nucleotides. Although the mature miRNA is very small, it is derived from a transcriptional product of a few hundred to a few thousand nucleotides. This process of maturation is known as miRNA biogenesis, extensively reviewed by Kim.5 Biogenesis of miR-143 and miR-145 is pictorially presented in Figure 1. Functionally, miRNAs are noncoding RNAs that negatively regulate gene expression. In the current, generally accepted model, they act mostly by inducing an inhibition of the translation of their target mRNAs, and, in a minority of cases, via their degradation.6,7 Very recently, however, Bartel's team challenged this view by showing that, in a vast majority of cases, mammalian microRNAs act by destabilizing their target mRNAs and decreasing their levels. …

Re-dicing the pancreatic β-cell: do microRNAs define cellular identity?

Re-dicing the pancreatic β-cell: do microRNAs define cellular identity?

Perturbations of MicroRNA Function in Mouse Dicer Mutants Produce Retinal Defects and Lead to Aberrant Axon Pathfinding at the Optic Chiasm

BackgroundDuring development axons encounter a variety of choice points where they have to make appropriate pathfinding decisions. The optic chiasm is a major decision point for retinal ganglion cell (RGC) axons en route to their target in order to ensure the correct wiring of the visual system. MicroRNAs (miRNAs) belong to the class of small non-coding RNA molecules and have been identified as important regulators of a variety of processes during embryonic development. However, their involvement in axon guidance decisions is less clear.Methodology/Principal FindingsWe report here that the early loss of Dicer, an essential protein for the maturation of miRNAs, in all cells of the forming retina and optic chiasm leads to severe phenotypes of RGC axon pathfinding at the midline. Using a conditional deletion approach in mice, we find in homozygous Dicer mutants a marked increase of ipsilateral projections, RGC axons extending outside the optic chiasm, the formation of a secondary optic tract and a substantial number of RGC axons projecting aberrantly into the contralateral eye. In addition, the mutant mice display a microphthalmia phenotype.ConclusionsOur work demonstrates an important role of Dicer controlling the extension of RGC axons to the brain proper. It indicates that miRNAs are essential regulatory elements for mechanisms that ensure correct axon guidance decisions at the midline and thus have a central function in the establishment of circuitry during the development of the nervous system.

Structural and biochemical insights into the dicing mechanism of mouse Dicer: A conserved lysine is critical for dsRNA cleavage

Dicer, an RNase III enzyme, initiates RNA interference by processing precursor dsRNAs into mature microRNAs and small-interfering RNAs. It is also involved in loading and activation of the RNA-induced silencing complex. Here, we report the crystal structures of a catalytically active fragment of mouse Dicer, containing the RNase IIIb and dsRNA binding domains, in its apo and Cd(2+)-bound forms, at 1.68- and 2.8-A resolution, respectively. Models of this structure with dsRNA reveal that a lysine residue, highly conserved in Dicer RNase IIIa and IIIb domains and in Drosha RNase IIIb domains, has the potential to participate in the phosphodiester bond cleavage reaction by stabilizing the transition state and leaving group of the scissile bond. Mutational and enzymatic assays confirm the importance of this lysine in dsRNA cleavage, suggesting that this lysine represents a conserved catalytic residue of Dicers. The structures also reveals a approximately 45-aa region within the RNase IIIb domain that harbors an alpha-helix at the N-terminal half and a flexible loop at the C-terminal half, features not present in previously reported structures of homologous RNase III domains from either bacterial RNase III enzymes or Giardia Dicer. N-terminal residues of this alpha-helix have the potential to engage in minor groove interaction with dsRNA substrates.

Molecular characterization of a mouse cDNA encoding Dicer, a ribonuclease III ortholog involved in RNA interference.

Members of the ribonuclease III superfamily of double-stranded(ds)-RNA-specific endoribonucleases participate in diverse cellular RNA maturation and degradation pathways. A recently identified eukaryotic RNase III family member, named "Dicer", functions in the RNA interference (RNAi) pathway by producing 21--23 bp dsRNAs which target the selective destruction of homologous RNAs. RNAi is operative in animals, plants, and fungi, where it is proposed to inhibit viral reproduction and retroposon movement, as well as to participate in developmental pathways. RNAi functions in mammalian cells, including mouse oocytes and embryos. This article reports the cDNA sequence characterization and expression analysis of the mouse Dicer ortholog. On the basis of the cDNA sequence, the Dicer polypeptide is 1906 amino acids and has a predicted molecular mass of 215 kDa. Mouse Dicer contains a DExH/DEAH helicase motif; a PAZ domain; a tandem repeat of RNase III catalytic domain sequences; and a dsRNA-binding motif. The Dicer gene maps to a single locus on the distal portion of mouse Chromosome (Chr) 12. The Dicer transcript is expressed from the embryonic through adult stages of development. The Dicer transcript is also present in a wide variety of adult mouse organs. The highly conserved set of functional domains and the occurrence of a single-copy gene strongly indicate that the encoded protein is the RNase III ortholog responsible for dsRNA processing in the RNAi pathway.