Dicer Activity Research Articles

Production of small RNAs by mammalian Dicer

MicroRNA (miRNA) and RNA interference (RNAi) pathways employ RNase III Dicer for the biogenesis of small RNAs guiding post-transcriptional repression. Requirements for Dicer activity differ in the two pathways. The biogenesis of miRNAs requires a single Dicer cleavage of a short hairpin precursor to produce a small RNA with a precisely defined sequence, while small RNAs in RNAi come from a processive cleavage of a long double-stranded RNA (dsRNA) into a pool of small RNAs with different sequences. While Dicer is generally conserved among eukaryotes, its substrate recognition, cleavage, and biological roles differ. In Metazoa, a single Dicer can function as a universal factor for RNAi and miRNA pathways or as a factor adapted specifically for one of the pathways. In this review, we focus on the structure, function, and evolution of mammalian Dicer. We discuss key structural features of Dicer and other factors defining Dicer substrate repertoire and biological functions in mammals in comparison with invertebrate models. The key for adaptation of Dicer for miRNA or RNAi pathways is the N-terminal helicase, a dynamically evolving Dicer domain. Its functionality differs between mammals and invertebrates: the mammalian Dicer is well adapted to produce miRNAs while its ability to support RNAi is limited.

Revealing a new activity of the human Dicer DUF283 domain in vitro.

The ribonuclease Dicer is a multidomain enzyme that plays a fundamental role in the biogenesis of small regulatory RNAs (srRNAs), which control gene expression by targeting complementary transcripts and inducing their cleavage or repressing their translation. Recent studies of Dicer’s domains have permitted to propose their roles in srRNA biogenesis. For all of Dicer’s domains except one, called DUF283 (domain of unknown function), their involvement in RNA substrate recognition, binding or cleavage has been postulated. For DUF283, the interaction with Dicer’s protein partners has been the only function suggested thus far. In this report, we demonstrate that the isolated DUF283 domain from human Dicer is capable of binding single-stranded nucleic acids in vitro. We also show that DUF283 can act as a nucleic acid annealer that accelerates base-pairing between complementary RNA/DNA molecules in vitro. We further demonstrate an annealing activity of full length human Dicer. The overall results suggest that Dicer, presumably through its DUF283 domain, might facilitate hybridization between short RNAs and their targets. The presented findings reveal the complex nature of Dicer, whose functions may extend beyond the biogenesis of srRNAs.

Dicer-independent RNA-directed DNA methylation in Arabidopsis.

RNA-directed DNA methylation (RdDM) is an important de novo DNA methylation pathway in plants. Small interfering RNAs (siRNAs) generated by Dicers from RNA polymerase IV (Pol IV) transcripts are thought to guide sequence-specific DNA methylation. To gain insight into the mechanism of RdDM, we performed whole-genome bisulfite sequencing of a collection of Arabidopsis mutants, including plants deficient in Pol IV (nrpd1) or Dicer (dcl1/2/3/4) activity. Unexpectedly, of the RdDM target loci that required Pol IV and/or Pol V, only 16% were fully dependent on Dicer activity. DNA methylation was partly or completely independent of Dicer activity at the remaining Pol IV- and/or Pol V-dependent loci, despite the loss of 24-nt siRNAs. Instead, DNA methylation levels correlated with the accumulation of Pol IV-dependent 25-50 nt RNAs at most loci in Dicer mutant plants. Our results suggest that RdDM in plants is largely guided by a previously unappreciated class of Dicer-independent non-coding RNAs, and that siRNAs are required to maintain DNA methylation at only a subset of loci.

Renal stromal miRNAs are required for normal nephrogenesis and glomerular mesangial survival.

MicroRNAs are small noncoding RNAs that post-transcriptionally regulate mRNA levels. While previous studies have demonstrated that miRNAs are indispensable in the nephron progenitor and ureteric bud lineage, little is understood about stromal miRNAs during kidney development. The renal stroma (marked by expression of FoxD1) gives rise to the renal interstitium, a subset of peritubular capillaries, and multiple supportive vascular cell types including pericytes and the glomerular mesangium. In this study, we generated FoxD1GC;Dicerfl/fl transgenic mice that lack miRNA biogenesis in the FoxD1 lineage. Loss of Dicer activity resulted in multifaceted renal anomalies including perturbed nephrogenesis, expansion of nephron progenitors, decreased renin-expressing cells, fewer smooth muscle afferent arterioles, and progressive mesangial cell loss in mature glomeruli. Although the initial lineage specification of FoxD1+ stroma was not perturbed, both the glomerular mesangium and renal interstitium exhibited ectopic apoptosis, which was associated with increased expression of Bcl2l11 (Bim) and p53 effector genes (Bax, Trp53inp1, Jun, Cdkn1a, Mmp2, and Arid3a). Using a combination of high-throughput miRNA profiling of the FoxD1+-derived cells and mRNA profiling of differentially expressed transcripts in FoxD1GC;Dicerfl/fl kidneys, at least 72 miRNA:mRNA target interactions were identified to be suppressive of the apoptotic program. Together, the results support an indispensable role for stromal miRNAs in the regulation of apoptosis during kidney development.

Context-dependent regulation of Dicer activity and small RNA production: Implications to oocyte-to-embryo transition

Cellular and molecular mechanisms that suppress small RNAs in oocytes while maintaining them in zygotes remain unknown. Signal-mediated regulation of small RNA biogenesis pathway is emerging as a theme for regulating small RNA production. We recently reported that ERK-mediated phosphorylation of Dicer, a central player in small RNA biogenesis, induced Dicer to move from the cytoplasm to the nucleus. Dicer phosphorylation inhibited its function, e.g., the production of 26G endo-siRNAs in the female germline. Moreover, our findings showed that the inhibition of Dicer function was necessary for normal progression of meiosis I and oogenesis, and that Dicer function had to be restored before fertilization for normal progression of embryogenesis. Thus, extracellular signal-dependent inhibition and then reactivation of Dicer is essential for oocyte-to-embryo transition. Strikingly, signal-induced Dicer translocation from the cytoplasm to nucleus is evolutionarily conserved from worm, flies, mice to humans thereby suggesting the ERK-mediated control of Dicer activity may be a generalized mechanism for regulating small RNA biogenesis.

Dysregulated miRNA biogenesis downstream of cellular stress and ALS-causing mutations: a new mechanism for ALS.

Interest in RNA dysfunction in amyotrophic lateral sclerosis (ALS) recently aroused upon discovering causative mutations in RNA-binding protein genes. Here, we show that extensive down-regulation of miRNA levels is a common molecular denominator for multiple forms of human ALS. We further demonstrate that pathogenic ALS-causing mutations are sufficient to inhibit miRNA biogenesis at the Dicing step. Abnormalities of the stress response are involved in the pathogenesis of neurodegeneration, including ALS. Accordingly, we describe a novel mechanism for modulating microRNA biogenesis under stress, involving stress granule formation and re-organization of DICER and AGO2 protein interactions with their partners. In line with this observation, enhancing DICER activity by a small molecule, enoxacin, is beneficial for neuromuscular function in two independent ALS mouse models. Characterizing miRNA biogenesis downstream of the stress response ties seemingly disparate pathways in neurodegeneration and further suggests that DICER and miRNAs affect neuronal integrity and are possible therapeutic targets.

Abstract A25: Heterozygous deletion of Dicer promotes anoikis and reduces pancreatic cancer metastasis in vivo

Abstract Previous studies have suggested that Dicer acts as a haploinsufficient tumor and metastasis suppressor. In contrast, studies of human pancreatic ductal adenocarcinoma (PDAC) tumors and cell lines demonstrate upregulation of miRNAs with progression. And yet, disagreement exists regarding which miRNA-target relationships are critical to tumor biology. We therefore sought to ascertain the effects of global miRNA suppression on pancreatic tumorigenesis using a conditional loss-of-function allele of Dicer in a mouse model of pancreatic cancer. Given the common observation of elevated miRNA expression in PDAC, we hypothesized that loss of one allele of Dicer would inhibit pancreatic tumorigenesis and prolong overall survival. Ptf1a-Cre-mediated deletion of Dicer in the pancreas demonstrated that homozygous loss is not tolerated, leading to progressive acinar cell atrophy as mice aged and suggesting that Dicer activity is required for the long-term viability of this cell type in the postnatal pancreas. However, mice with heterozygous deletion of Dicer had developmentally normal pancreata that were maintained into adulthood. To assess the impact of Dicer gene deletion on pancreatic tumorigenesis in vivo, we used the RCAS-TVA pancreatic cancer model incorporating polyoma virus middle T antigen (PyMT) expression and p53 gene deletion. We found that heterozygous deletion of Dicer did not impact overall animal survival; primary tumor size, number, or burden; or tumor histology (acinar, ductal, or poorly differentiated carcinomas). However, loss of one copy of Dicer robustly inhibits metastasis of PyMT-driven pancreatic tumors, with metastases seeding from 12.5% of DicerFL/WT tumors versus 54.5% of DicerWT/WT tumors (p<0.05). Intriguingly, cell lines derived from DicerWT/WT and DicerFL/WT tumors also display no differences in their proliferative rate, migration, invasion, soft agar colony formation, or survival following serum-starvation. However, DicerFL/WT cancer cell lines exhibit markedly increased sensitivity to cell death from loss of attachment (anoikis) compared to DicerWT/WT lines (50% survival vs 76% survival at 24 hours; p<0.01). This result provides a potential explanation for the reduced metastasis seen with DicerFL/WT tumors in our mouse model, and it invites further investigation into the precise miRNAs responsible for this phenotype. Importantly, our findings are consistent with observations in human pancreatic cancer, where upregulation of oncogenic miRNAs is commonly seen in late-stage disease. However, our data are in disagreement with published literature implicating Dicer as a tumor suppressor whose expression is decreased during carcinoma progression in other tumor contexts. This discrepancy highlights the importance of cellular context in understanding miRNA biology and the need for more thorough in vivo genetic experimentation to dissect the role of miRNAs in pancreatic carcinogenesis. Citation Format: Brian Quattrochi, Brian C. Lewis. Heterozygous deletion of Dicer promotes anoikis and reduces pancreatic cancer metastasis in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A25.

Morphine Promotes Astrocyte-Preferential Differentiation of Mouse Hippocampal Progenitor Cells via PKCε-Dependent ERK Activation and TRBP Phosphorylation.

Previously we have shown that morphine regulates adult neurogenesis by modulating miR-181a maturation and subsequent hippocampal neural progenitor cell (NPC) lineages. Using NPCs cultured from PKCε or β-arrestin2 knockout mice and the MAPK/ERK kinase inhibitor U0126, we demonstrate that regulation of NPC differentiation via the miR-181a/Prox1/Notch1 pathway exhibits ligand-dependent selectivity. In NPCs, morphine and fentanyl activate ERK via the PKCε- and β-arrestin-dependent pathways, respectively. After fentanyl exposure, the activated phospho-ERK translocates to the nucleus. Conversely, after morphine treatment, phospho-ERK remains in the cytosol and is capable of phosphorylating TAR RNA-binding protein (TRBP), a cofactor of Dicer. This augments Dicer activity and promotes the maturation of miR-181a. Furthermore, using NPCs transfected with wild-type TRBP, SΔA, and SΔD TRBP mutants, we confirmed the crucial role of TRBP phosphorylation in Dicer activity, miR-181a maturation, and finally the morphine-induced astrocyte-preferential differentiation of NPCs. Thus, morphine modulates the lineage-specific differentiation of NPCs by PKCε-dependent ERK activation with subsequent TRBP phosphorylation and miR-181a maturation.

Cross-species comparative analysis of Dicer proteins during Sindbis virus infection.

In plants and invertebrates RNA silencing is a major defense mechanism against virus infections. The first event in RNA silencing is dicing of long double stranded RNAs into small interfering RNAs (siRNAs). The Dicer proteins involved in this process are phylogenetically conserved and have the same domain organization. Accordingly, the production of viral derived siRNAs has also been observed in the mouse, but only in restricted cell types. To gain insight on this restriction, we compare the dicing activity of human Dicer and fly Dicer-2 in the context of Sindbis virus (SINV) infection. Expression of human Dicer in flies inefficiently rescues the production of viral siRNAs but confers some protection against SINV. Conversely, expression of Dicer-2 in human cells allows the production of viral 21 nt small RNAs. However, this does not confer resistance to viral infection, but on the contrary results in stronger accumulation of viral RNA. We further show that Dicer-2 expression in human cells perturbs interferon (IFN) signaling pathways and antagonizes protein kinase R (PKR)-mediated antiviral immunity. Overall, our data suggest that a functional incompatibility between the Dicer and IFN pathways explains the predominance of the IFN response in mammalian somatic cells.

Increased Viral Dissemination in the Brain and Lethality in MCMV-Infected, Dicer-Deficient Neonates.

Among Herpesviruses, Human Cytomegalovirus (HCMV or HHV-5) represents a major threat during congenital or neonatal infections, which may lead to encephalitis with serious neurological consequences. However, as opposed to other less prevalent pathogens, the mechanisms and genetic susceptibility factors for CMV encephalitis are poorly understood. This lack of information considerably reduces the prognostic and/or therapeutic possibilities. To easily monitor the effects of genetic defects on brain dissemination following CMV infection we used a recently developed in vivo mouse model based on the neonatal inoculation of a MCMV genetically engineered to express Luciferase. Here, we further validate this protocol for live imaging, and demonstrate increased lethality associated with viral infection and encephalitis in mutant mice lacking Dicer activity. Our data indicate that miRNAs are important players in the control of MCMV pathogenesis and suggest that miRNA-based endothelial functions and integrity are crucial for CMV encephalitis.

A New Tool for in vivo Manipulation of Brain microRNA Levels: The Work of Smalheiser et al. (2014)

A New Tool for in vivo Manipulation of Brain microRNA Levels: The Work of Smalheiser et al. (2014)

The many faces of Dicer: the complexity of the mechanisms regulating Dicer gene expression and enzyme activities.

There is increasing evidence indicating that the production of small regulatory RNAs is not the only process in which ribonuclease Dicer can participate. For example, it has been demonstrated that this enzyme is also involved in chromatin structure remodelling, inflammation and apoptotic DNA degradation. Moreover, it has become increasingly clear that cellular transcript and protein levels of Dicer must be strictly controlled because even small changes in their accumulation can initiate various pathological processes, including carcinogenesis. Accordingly, in recent years, a number of studies have been performed to identify the factors regulating Dicer gene expression and protein activity. As a result, a large amount of complex and often contradictory data has been generated. None of these data have been subjected to an exhaustive review or critical discussion. This review attempts to fill this gap by summarizing the current knowledge of factors that regulate Dicer gene transcription, primary transcript processing, mRNA translation and enzyme activity. Because of the high complexity of this topic, this review mainly concentrates on human Dicer. This review also focuses on an additional regulatory layer of Dicer activity involving the interactions of protein and RNA factors with Dicer substrates.

Regulatory networks between neurotrophins and miRNAs in brain diseases and cancers.

Neurotrophins are involved in many physiological and pathological processes in the nervous system. They regulate and modify signal transduction, transcription and translation in neurons. It is recently demonstrated that the neurotrophin expression is regulated by microRNAs (miRNAs), changing our views on neurotrophins and miRNAs. Generally, miRNAs regulate neurotrophins and their receptors in at least two ways: (1) miRNAs bind directly to the 3′ untranslated region (UTR) of isoform-specific mRNAs and post-transcriptionally regulate their expression; (2) miRNAs bind to the 3′ UTR of the regulatory factors of neurotrophins and regulate their expression. On the other hand, neurotrophins can regulate miRNAs. The results of BNDF research show that neurotrophins regulate miRNAs in at least three ways: (1) ERK stimulation enhances the activation of TRBP (HIV-1 TAR RNA-binding protein) and Dicer, leading to the upregulation of miRNA biogenesis; (2) ERK-dependent upregulation of Lin28a (RNA-binding proteins) blocks select miRNA biogenesis; (3) transcriptional regulation of miRNA expression through activation of transcription factors, including CREB and NF-κB. These regulatory processes integrate positive and negative regulatory loops in neurotrophin and miRNA signaling pathways, and also expand the function of neurotrophins and miRNAs. In this review, we summarize the current knowledge of the regulatory networks between neurotrophins and miRNAs in brain diseases and cancers, for which novel cutting edge therapeutic, delivery and diagnostic approaches are emerging.

Dicer Switch

ERK-mediated phosphorylation alters the localization and activity of Dicer during oogenesis in Caenorhabditis elegans .

Dicer nuclease-promoted production of Let7a-1 microRNA is enhanced in the presence of tryptophan-containing amphiphilic peptides.

A library of Trp-containing amphiphilic peptides was synthesized and screened for the ability to bind to pre-miRNA targets. Two members of this family, peptides Ac-WKKLLKWLKKLLKLAG-NH2 (2 b) and Ac-WKKLLKWLKKLLDabLAG-NH2 (4 b) were found to have nanomolar binding affinities to pre-let7a-1. Peptides 2 b and 4 b caused an increase in the in vitro Dicer cleavage of pre-let7a-1. This observation was confirmed by a cell-based assay, the results of which show an up to 50 % increase in Dicer activity. Enhanced expression of let7a-1 promoted by the peptides results in specific reductions of target mRNAs. The results of a microarray study show that lower amount of fluctuating genes are generated in the presence of 2 b or 4 b, relative to that from exogenous delivery of let7a-1. Because peptides 2 b and 4 b promote enhanced formation of mature let7a-1 only at the endogenous miRNA level, this specifically controls genes related to let7a-1.

A MicroRNA Processing Defect in Smokers' Macrophages Is Linked to SUMOylation of the Endonuclease DICER

Despite the fact that alveolar macrophages play an important role in smoking-related disease, little is known about what regulates their pathophysiologic phenotype. Evaluating smoker macrophages, we found significant down-regulation of multiple microRNAs (miRNAs). This work investigates the hypothesis that cigarette smoke alters mature miRNA expression in lung macrophages by inhibiting processing of primary miRNA transcripts. Studies on smoker alveolar macrophages showed a defect in miRNA maturation. Studies on the miRNA biogenesis machinery led us to focus on the cytosolic RNA endonuclease, DICER. DICER cleaves the stem-loop structure from pre-miRNAs, allowing them to dissociate into their mature 20-22-nucleotide single-stranded form. DICER activity assays confirmed impaired DICER activity following cigarette smoke exposure. Further protein studies demonstrated a decreased expression of the native 217-kDa form of DICER and an accumulation of high molecular weight forms with cigarette smoke exposure. This molecular mass shift was shown to contain SUMO moieties and could be blocked by silencing RNA directed at the primary SUMOylating ligase, Ubc9. In determining the cigarette smoke components responsible for changes in DICER, we found that N-acetylcysteine, an antioxidant and anti-aldehyde, protected DICER protein and activity from cigarette smoke extract. This massive down-regulation of miRNAs (driven in part by alterations in DICER) may be an important regulator of the disease-promoting macrophage phenotype found in the lungs of smokers.

Trans-activation Response (TAR) RNA-binding Protein 2 Is a Novel Modulator of Transient Receptor Potential Canonical 4 (TRPC4) Protein

TRPC4 proteins function as Ca(2+) conducting, non-selective cation channels in endothelial, smooth muscle, and neuronal cells. To further characterize the roles of TRPC4 in vivo, detailed information about the molecular composition of native channel complexes and their association with cellular signaling networks is needed. Therefore, a mouse brain cDNA library was searched for novel TRPC4-interacting proteins using a modified yeast two-hybrid assay. This screen identified Trans-activation Response RNA-binding protein 2 (Tarpb2), a protein that recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Tarbp2 was found to bind to the C terminus of TRPC4 and TRPC5 and to modulate agonist-dependent TRPC4-induced Ca(2+) entry. A stretch of basic residues within the Tarbp2 protein is required for these actions. Tarbp2 binding to and modulation of TRPC4 occurs in the presence of endogenously expressed Dicer but is no longer detectable when the Dicer cDNA is overexpressed. Dicer activity in crude cell lysates is increased in the presence of Ca(2+), most probably by Ca(2+)-dependent proteolytic activation of Dicer. Apparently, Tarbp2 binding to TRPC4 promotes changes of cytosolic Ca(2+) and, thereby, leads to a dynamic regulation of Dicer activity, essentially at low endogenous Dicer concentrations.

Mutant p53 Regulates Dicer through p63-dependent and -independent Mechanisms to Promote an Invasive Phenotype

The control and processing of microRNAs (miRs) is critical in the regulation of all cellular responses. Previous studies have suggested that a reduction in the expression of certain miRs, or an overall decrease in miR processing through the partial depletion of Dicer, can promote enhanced metastatic potential. We show here that Dicer depletion can promote the invasive behavior of cells that is reflected in enhanced recycling and activation of the growth factor receptors Met and EGF receptor. These responses are also seen in response to the expression of tumor-derived mutant p53s, and we show that mutant p53 can down-regulate Dicer expression through both direct inhibition of the TAp63-mediated transcriptional activation of Dicer and a TAp63-independent control of Dicer protein expression. Our results delineate a clear relationship between mutant p53, TAp63, and Dicer that might contribute to the metastatic function of mutant p53 but, interestingly, also reveal TAp63-independent functions of mutant p53 in controlling Dicer activity.

MicroRNA biogenesis: dicing assay.

RNA-induced silencing complex is the cytoplasmic effector machine of the microRNA (miRNA) pathway and contains a single-stranded miRNA guiding it to its target mRNAs. The biogenesis of mature miRNAs is a regulatory process achieved by complex machinery composed of few protein components, among which the ribonuclease III Dicer plays a central role. Dicer is essential for miRNA maturation and catalyzes one of the rate-limiting steps of miRNA production. In this chapter, we describe a protocol to study the catalytic activity of Dicer in cell extracts by measuring their ability to process precursor RNA (pre-miRNAs) into functional mature miRNAs. Impairment of the miRNA biogenesis machinery due to loss-of-function mutations in effectors of the pathway such as Dicer has been demonstrated before. Dicing assay can be used in cancer to assess Dicer enzymatic activity compared with healthy controls. Therefore, this experimental approach is likely to be useful to researchers investigating the main steps of miRNA biogenesis and function in human health and diseases.

MiRNA regulatory mechanism in tumor initiation and progression

MiRNAs can function as oncogenes or tumor suppressor genes. The abnormal expression of miRNAs leads to tumor malignant phenotypes, such as cell proliferation, apoptosis, invasion and metastasis, through which it is involved in the process of tumor initiation, progression and transcriptional regulation network. Therefore, it is important to clarify the mechanism of miRNA involved in the process of tumor initiation and progression. MiRNA regulation mechanism in tumor initiation and progression includes one-to-many and many-to-one regulation between TFto- miRNA and miRNA-to-target gene, which increases the complexity of miRNA regulation, thus affecting the biological behavior of the tumor, The expression and activity of Drosha and Dicer in the process of miRNA affect the synthesis of mature miRNA and involve in the process of tumor initiation and progression; ceRNA may bind with miRNA by competing with miRNA targeting genes and affect biological function of miRNA as miRNA inhibitor. Therefore the abnormal expression and structure of ceRNA is an important molecular mechanism of tumor initiation and progression. This complicated regulation network comprised by multi-dimensional regulation model and specific regulation of tumor initiation and progression provides impetus to exploring the functional restoration of miRNA as a novel target for cancer diagnosis and therapy.