Evolutionary conservation of a unique amino acid sequence in human DICER protein essential for binding to Argonaute family proteins

The Argonaute family and DICER proteins are major key proteins involved in the RNA-mediated gene silencing mechanism of various species. In this mechanism, cleavage of messenger RNAs (mRNA) or suppression of mRNA translation takes place via small RNAs that are uniquely processed by DICER. Previously, we demonstrated that human Argonaute family proteins bind to DICER. In this study, we identified a unique amino acid sequence of 127 amino acids in the RIBOc-A domain of human DICER protein as a "binding site" to Argonaute proteins. Comparative genomics analysis revealed that this unique amino acid sequence is highly conserved in the vertebrates, but not found in the non-vertebrate species. Significant difference in the RIBOc-A domain of DICER protein between vertebrate and non-vertebrate species may help exploring the functional complexity in the RNA-mediated gene silencing mechanism.     

Nuclear pore complex protein mediated nuclear localization of dicer protein in human cells

Human DICER1 protein cleaves double-stranded RNA into small sizes, a crucial step in production of single-stranded RNAs which are mediating factors of cytoplasmic RNA interference. Here, we clearly demonstrate that human DICER1 protein localizes not only to the cytoplasm but also to the nucleoplasm. We also find that human DICER1 protein associates with the NUP153 protein, one component of the nuclear pore complex. This association is detected predominantly in the cytoplasm but is also clearly distinguishable at the nuclear periphery. Additional characterization of the NUP153-DICER1 association suggests NUP153 plays a crucial role in the nuclear localization of the DICER1 protein.     

A critical switch in the enzymatic properties of the Cid1 protein deciphered from its product-bound crystal structure

The addition of uridine nucleotide by the poly(U) polymerase (PUP) enzymes has a demonstrated impact on various classes of RNAs such as microRNAs (miRNAs), histone-encoding RNAs and messenger RNAs. Cid1 protein is a member of the PUP family. We solved the crystal structure of Cid1 in complex with non-hydrolyzable UMPNPP and a short dinucleotide compound ApU. These structures revealed new residues involved in substrate/product stabilization. In particular, one of the three catalytic aspartate residues explains the RNA dependence of its PUP activity. Moreover, other residues such as residue N165 or the β-trapdoor are shown to be critical for Cid1 activity. We finally suggest that the length and sequence of Cid1 substrate RNA influence the balance between Cid1''s processive and distributive activities. We propose that particular processes regulated by PUPs require the enzymes to switch between the two types of activity as shown for the miRNA biogenesis where PUPs can either promote DICER cleavage via short U-tail or trigger miRNA degradation by adding longer poly(U) tail. The enzymatic properties of these enzymes may be critical for determining their particular function in vivo.     

miSSING LINKS: miRNAs and plant development

The discovery of hundreds of plant micro RNAs (miRNAs) has triggered much speculation about their potential roles in plant development. The search for plant genes involved in miRNA processing has revealed common factors such as DICER, and new molecules, including HEN1. Progress is also being made toward identifying miRNA target genes and understanding the mechanisms of miRNA-mediated gene regulation in plants. This work has lead to a reexamination of many previously characterized mutations that are now known to affect components or targets of miRNA-mediated pathways.     

Neuroprotective effects of valproic acid against hemin toxicity: Possible involvement of the down-regulation of heme oxygenase-1 by regulating ubiquitin–proteasomal pathway

During hemorrhagic stroke induced by intracerebral hemorrhage (ICH), brain injury occurs from the deleterious actions of hemoglobin byproducts; induction of heme oxygenase-1 (HO-1) also plays a critical role in the neurotoxicity in ICH. Valproic acid (VPA), which is a commonly used drug in the treatment of epilepsy, has been reported to have neuroprotective effects against various neuronal insults including ischemic stroke. We investigated the effect of VPA on HO-1-mediated neurotoxicity in an experimental model of ICH. We investigated the effects of VPA on HO-1 protein in primary cortical neurons: (1) the expression levels of HO-1 mRNA and protein measured by RT-PCR and Western blotting; (2) the cell viability and ROS generation by MTT reduction assay and ROS measurement; (3) the signal pathway regulated by VPA using IP-Western blotting; (4) the effects of VPA on hemin-induced cell death by hemin microinjection and immunohistochemistry in vivo. VPA treatment partially blocked cell death induced by hemin, which is released from hemoglobin during ICH, both in rat primary cortical neurons and rat brain. Treatment of VPA significantly decreased the expression of HO-1 protein both in vitro and in vivo. Hemin treatment induced HO-1 protein expression and this was partially blocked by pretreatment with VPA, which might be mediated by increased ubiquitination and degradation of HO-1 via ERK1/2 and JNK activation in primary cortical neurons. Our results indicate that VPA inhibits hemin toxicity by downregulating HO-1 protein expression, and provide a therapeutic strategy to attenuate intracerebral hemorrhagic injury.     

DICER-dependent biogenesis of let-7 miRNAs affects human cell response to DNA damage via targeting p21/p27

Recently, it was reported that knockdown of DICER reduced the ATM-dependent DNA damage response and homologous recombination repair (HRR) via decreasing DICER-generated small RNAs at the damage sites. However, we found that knockdown of DICER dramatically increased cell resistance to camptothecin that induced damage required ATM to facilitate HRR. This phenotype is due to a prolonged G1/S transition via decreasing DICER-dependent biogenesis of miRNA let-7, which increased the p21^Waf1/Cip1/p27^Kip1 levels and resulted in decreasing the HRR efficiency. These results uncover a novel function of DICER in regulating the cell cycle through miRNA biogenesis, thus affecting cell response to DNA damage.     

Role of Dicer1 in thyroid cell proliferation and differentiation

DICER1 plays a central role in the biogenesis of microRNAs and it is important for normal development. Altered microRNA expression and DICER1 dysregulation have been described in several types of tumors, including thyroid carcinomas. Recently, our group identified a new somatic mutation (c.5438A>G; E1813G) within DICER1 gene of an unknown function. Herein, we show that DICER1 is overexpressed, at mRNA level, in a significant-relative number of papillary (70%) and anaplastic (42%) thyroid carcinoma samples, whereas is drastically downregulated in all the analyzed human thyroid carcinoma cell lines (TPC-1, BCPAP, FRO and 8505c) in comparison with normal thyroid tissue samples. Conversely, DICER1 is downregulated, at protein level, in PTC in comparison with normal thyroid tissues. Our data also reveals that DICER1 overexpression positively regulates thyroid cell proliferation, whereas its silencing impairs thyroid cell differentiation. The expression of DICER1 gene mutation (c.5438A>G; E1813G) negatively affects the microRNA machinery and cell proliferation as well as upregulates DICER1 protein levels of thyroid cells but has no impact on thyroid differentiation. In conclusion, DICER1 protein is downregulated in papillary thyroid carcinomas and affects thyroid proliferation and differentiation, while DICER1 gene mutation (c.5438A>G; E1813G) compromises the DICER1 wild-type-mediated microRNA processing and cell proliferation.     

Deficiency of DICER reduces the invasion ability of trophoblasts and impairs the pro‐angiogenic effect of trophoblast‐derived microvesicles

DICER is a key rate‐limiting enzyme in the canonical miRNAs biogenesis pathway, and DICER and DICER‐dependent miRNAs have been proved to play essential roles in many physiological and pathological processes. However, whether DICER is involved in placentation has not been studied. Successful spiral artery remodelling is one of the key milestones during placentation, which depends mostly on the invasion of trophoblasts and the crosstalk between trophoblasts and endothelial cells. In the present study, we show that DICER knockdown impairs the invasion ability of both primary extravillous trophoblasts (EVT) and HTR8/SVneo (HTR8) cell lines. The decreased invasion of HTR8 cells upon DICER knockdown (sh‐Dicer) was partly due to the up‐regulation of miR‐16‐2‐3p, which led to a reduced expression level of the collagen type 1 alpha 2 chain (COL1A2) protein. Moreover, microvesicles (MVs) can be secreted by HTR8 cells and promote the tube formation ability of human umbilical cord vein endothelial cells (HUVECs). However, conditioned medium and MVs derived from sh‐Dicer HTR8 cells have an anti‐angiogenic effect, due to reduced angiogenic factors and increased anti‐angiogenic miRNAs (including let‐7d, miR‐1‐6‐2 and miR‐15b), respectively. In addition, reduced protein expression of DICER is found in PE placenta by immunoblotting and immunohistochemistry. In summary, our study uncovered a novel DICER‐miR‐16‐2‐COL1A2 mediated pathway involved in the invasion ability of EVT, and DICER‐containing MVs mediate the pro‐angiogenic effect of trophoblast‐derived conditioned medium on angiogenesis, implying the involvement of DICER in the pathogenesis of PE.     

Mutagens interfere with microRNA maturation by inhibiting DICER. An in silico biology analysis

Exposure to environmental mutagens results in alteration of microRNA expression mainly oriented towards down-regulation, as typically observed in cigarette smoke. However, the molecular mechanism triggering this event is still unknown. To shed light on this issue, we developed an 'in silico' analysis testing 25 established environmental mutagens (polycyclic aromatic hydrocarbons, heterocyclic compounds, nitrosoamines, morpholine, ethylnitrosurea, benzene derivatives, hydroxyl amines, alkenes) for their potential to interfere with the function of DICER, the enzyme involved in the cytoplasmic phase of microRNA maturation. In order to analyse the binding affinity between DICER and each mutagen, the three-dimensional bioinformatic structures of DICER-RNase III domains and of mutagens have been constructed. The binding affinity of mutagens for each DICER's RNase III domain was estimated by calculating the global contact-energy and the number of intermolecular contacts. These two parameters reflect the stability of the DICER-mutagen complexes. All the 25 mutagens tested form stable complexes with DICER, 20 of which form a complex with DICER A domain, that is more stable than those formed by DICER with its natural substrate, i.e. double strand short RNAs. These mutagens are benzo(a)pyrene diol epoxide, nitroimidazoles, fluorenes, naphthalene, morpholine, stilbenes, hydroxylamines, fecapentenes. In the case of exposure to mutagen mixtures (benzo(a)pyrene-diolepoxide and 4-acetylaminostilbene), synergistic or less than addictive effects occur depending on the docking order of the compounds. A group of 8 mutagens with the highest ability to interfere with this DICER function, was identified by hierarchical cluster analysis. This group included 1-ethyl-1-nitrosourea and 4-nitrosomorpholine. Herein, presented data support the view that mutagens interfere with microRNA maturation by binding DICER. This finding sheds light on a new epigenetic mechanism exerted by environmental mutagens in inducing cell damage.     

Two-step cleavage of hairpin RNA with 5' overhangs by human DICER

Background  

DICER is an RNase III family endoribonuclease that processes precursor microRNAs (pre-miRNAs) and long double-stranded RNAs, generating microRNA (miRNA) duplexes and short interfering RNA duplexes with 20~23 nucleotides (nts) in length. The typical form of pre-miRNA processed by the Drosha protein is a hairpin RNA with 2-nt 3' overhangs. On the other hand, production of mature miRNA from an endogenous hairpin RNA with 5' overhangs has also been reported, although the mechanism for this process is unknown.     

Differentiation of rat adipose tissue-derived stem cells into neuron-like cells by valproic acid,a histone deacetylase inhibitor

Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to modulate the neuronal differentiation of adipose tissue-derived stem cells (ASCs) in humans and dogs. However, controversy exists as to whether VPA really acts as an inducer of neuronal differentiation of ASCs. The present study aimed to elucidate the effect of VPA in neuronal differentiation of rat ASCs. One or three days of pretreatment with VPA (2 mM) followed by neuronal induction enhanced the ratio of immature neuron marker βIII-tubulin-positive cells in a time-dependent manner, where the majority of cells also had a positive signal for neurofilament medium polypeptide (NEFM), a mature neuron marker. RT-PCR analysis revealed increases in the mRNA expression of microtubule-associated protein 2 (MAP2) and NEFM mature neuron markers, even without neuronal induction. Three-days pretreatment of VPA increased acetylation of histone H3 of ASCs as revealed by immunofluorescence staining. Chromatin immunoprecipitation assay also showed that the status of histone acetylation at H3K9 correlated with the gene expression of TUBB3 in ASCs by VPA. These results indicate that VPA significantly promotes the differentiation of rat ASCs into neuron-like cells through acetylation of histone H3, which suggests that VPA may serve as a useful tool for producing transplantable cells for future applications in clinical treatments.     

Arabidopsis DRB4 protein in antiviral defense against Turnip yellow mosaic virus infection

RNA silencing is an important antiviral mechanism in diverse eukaryotic organisms. In Arabidopsis DICER‐LIKE 4 (DCL4) is the primary antiviral Dicer, required for the production of viral small RNAs from positive‐strand RNA viruses. Here, we showed that DCL4 and its interacting partner dsRNA‐binding protein 4 (DRB4) participate in the antiviral response to Turnip yellow mosaic virus (TYMV), and that both proteins are required for TYMV‐derived small RNA production. In addition, our results indicate that DRB4 has a negative effect on viral coat protein accumulation. Upon infection DRB4 expression was induced and DRB4 protein was recruited from the nucleus to the cytoplasm, where replication and translation of viral RNA occur. DRB4 was associated with viral RNA in vivo and directly interacted in vitro with a TYMV RNA translational enhancer, raising the possibility that DRB4 might repress viral RNA translation. In plants the role of RNA silencing in viral RNA degradation is well established, but its potential function in the regulation of viral protein levels has not yet been explored. We observed that severe infection symptoms are not necessarily correlated with enhanced viral RNA levels, but might be caused by elevated accumulation of viral proteins. Our findings suggest that the control of viral protein as well as RNA levels might be important for mounting an efficient antiviral response.