• Chronic stress
• Cyclic GMP-AMP synthase
• Endogenous retrovirus
• Immuno-inflammation
• Microglia
• Negative emotional behavior
• Stimulator of interferon genes

• RNA Interference
• RNAi Therapeutics
• RNA, Small Interfering/genetics
• RNA, Small Interfering/therapeutic use
• MicroRNAs/genetics
• MicroRNAs/therapeutic use
• MicroRNAs/metabolism
• Bioengineering
• Biological Products

• P30 CA093373 NCI NIH HHS
• R01 CA225958 NCI NIH HHS
• T32 GM099608 NIGMS NIH HHS
• R35 GM140835 NIGMS NIH HHS
• R01 CA253230 NCI NIH HHS

• Bioengineering
• Biotechnology
• MicroRNA
• Noncoding RNA
• siRNA

• Bacteria/genetics
• Bacteria/metabolism
• Bioengineering
• Fermentation
• RNA, Untranslated/biosynthesis
• RNA, Untranslated/genetics

• R01 CA225958 NCI NIH HHS
• R01 GM113888 NIGMS NIH HHS
• R01GM113888 NIGMS NIH HHS
• R01CA225958 NCI NIH HHS

Dicer is a 219-kDa protein that plays key roles in gene regulation, particularly as the ribonuclease III enzyme responsible for cleaving precursor miRNA substrates. Its enzymatic activity is highly regulated by protein factors, and this regulation can impact on the levels of miRNAs and modulate the behavior of a cell. To better understand the underlying mechanisms of regulation, detailed enzymatic and structural characterization of Dicer are needed. However, these types of studies generally require several milligrams of recombinant protein, and efficient preparation of such quantities of pure human Dicer remains a challenge. To prepare large quantities of human Dicer, we have optimized transfection in HEK293-6E cells grown in suspension and streamlined a purification procedure.

Transfection conditions were first optimized to achieve expression levels between 10 and 18 mg of recombinant Dicer per liter of culture. A three-step purification protocol was then developed that yields 4–9 mg of purified Dicer per liter of culture in a single day. From SEC-MALS/RI analysis and negative stain TEM, we confirmed that the purified protein is monomerically pure ( ≥ 98%) and folds with the characteristic L-shape geometry. Using an electrophoretic mobility shift assay, a dissociation constant (K_d) of 5 nM was measured for Dicer binding to pre-let-7a-1, in agreement with previous reports. However, when probing the cleavage activity of Dicer for pre-let-7a-1, we measured k_cat (7.2 ± 0.5 min^− 1) and K_M (1.2 ± 0.3 μM) values that are much higher than previously reported due to experimental conditions that better respect the steady-state assumption.

The expression and purification protocols described here provide high yields of monomerically pure and active human Dicer. Cleavage studies of a pre-let-7 substrate with this purified Dicer reveal higher k_cat and K_M values than previously reported and support the current view that conformational changes are associated with substrate binding. Large quantities of highly pure Dicer will be valuable for future biochemical, biophysical and structural investigations of this key protein of the miRNA pathway.

The online version of this article (10.1186/s12896-018-0485-3) contains supplementary material, which is available to authorized users.

• Dicer binding
• Dicer cleavage assay
• Dicer expression
• Dicer purification
• Human HEK293-EBNA1 cells
• Mammalian cell suspension culture
• Pre-let-7
• Pre-miRNA

• Animals
• Antigens, Viral/immunology
• Arthropod Proteins/genetics
• Arthropod Proteins/metabolism
• Gene Expression Regulation
• Immunity, Innate
• Interferon Regulatory Factors/metabolism
• Interferons/metabolism
• Pathogen-Associated Molecular Pattern Molecules/immunology
• Penaeidae/immunology
• RNA, Double-Stranded/immunology
• RNA, Small Interfering/genetics
• Ribonuclease III/genetics
• Ribonuclease III/metabolism
• Signal Transduction
• Toll-Like Receptor 3/genetics
• Toll-Like Receptor 3/metabolism
• Virus Diseases/immunology

Although both long and micro RNAs are emerging as important functional components in colorectal cancer (CRC) progression and metastasis, the mechanism of their interaction remains poorly understood. CCAT2 (Colon cancer-associated transcript-2), a long noncoding RNA (lncRNA), has been reported to be over-expressed in CRC and is found to promote tumor growth. miRNAs, a class of naturally occurring short RNAs negatively control the expression of target genes by cleaving mRNA or through translation repression. Recently, we reported that miR-145 and miR-21 cooperate to regulate colon cancer stem cell (CSC) proliferation and differentiation. Considering that CCAT2 is mainly located in the nucleus and miRNA maturation process begins in the nucleus, we hypothesize that CCAT2 selectively blocks miR-145 maturation process, resulting in decreased mature miR-145 affecting colon CSC proliferation and differentiation.

The levels of CCAT2 were manipulated by transfection of CCAT2 expression plasmid or knockdown by siRNA or by CRISPR/Cas9. Quantitative RT-PCR was performed to examine the expression of CCAT2 and pri-, pre- and mature miR-145/21. Fluorescence in situ hybridization (FISH) was used to visualize CCAT2 in the cells. In vitro processing of pri-miRNA-145 was performed using T7 RNA polymerase and recombinant human Dicer.

We have observed that modulated expression of CCAT2 regulates the expression of miR-145 in colon cancer HCT-116 and HT-29 cells. Knockout of CCAT2 increases miR-145 and negatively regulates miR-21 in HCT-116 cells, impairs proliferation and differentiation. In contrast, stable up-regulation of CCAT2 decreases mature miR-145 and increases the expression of several CSC markers in colon cancer cells. We have also observed that CCAT2 is enriched in the nucleus and correlates with the expression of pre-miR-145 but not pre-miR-21 in HCT-116 cells. These results indicate CCAT2 selectively blocks miR-145 maturation by inhibiting pre-miR-145 export to cytoplasm. Further, we revealed that CCAT2 blocks cleavage of pre-miR-145 by Dicer in vitro.

Our results identify CCAT2 as a negative regulator of miRNA-145 biogenesis, and expose a novel mechanism of lncRNA-miRNA crosstalk.

The online version of this article (10.1186/s12943-017-0725-5) contains supplementary material, which is available to authorized users.

• cancer stem cells
• lncRNA-miRNA crosstalk
• miRNA biogenesis

• DCL
• Dicer
• DsRNA-binding protein
• MicroRNA
• Small RNA
• Small interfering RNA

• MicroRNAs/genetics
• MicroRNAs/metabolism
• Plant Proteins/genetics
• Plant Proteins/metabolism
• Plants/genetics
• Plants/metabolism
• RNA, Double-Stranded/genetics
• RNA, Double-Stranded/metabolism
• RNA, Plant/genetics
• RNA, Plant/metabolism
• RNA, Small Interfering/genetics
• RNA, Small Interfering/metabolism
• Ribonuclease III/genetics
• Ribonuclease III/metabolism

• Blotting, Western
• Cells, Cultured
• Fetal Proteins/metabolism
• Flow Cytometry
• Formins
• Human Umbilical Vein Endothelial Cells/metabolism
• Human Umbilical Vein Endothelial Cells/microbiology
• Humans
• Listeria monocytogenes
• Nuclear Proteins/metabolism
• Phagocytosis/physiology
• Polymerase Chain Reaction
• Proteins/metabolism

• T32 GM007365 NIGMS NIH HHS
• R01 AI036929 NIAID NIH HHS
• P50 GM107615 NIGMS NIH HHS
• S10 RR027431 NCRR NIH HHS
• R37 AI036929 NIAID NIH HHS
• Howard Hughes Medical Institute
• National Institute of Allergy and Infectious Diseases

• Computational modeling
• Extracellular matrix
• Immune checkpoint therapy
• Interstitial and transvascular transport
• Intracellular trafficking
• Nanoparticles
• Spatiokinetics
• Tumor microenvironment

• Animals
• Antineoplastic Agents/administration & dosage
• Antineoplastic Agents/pharmacokinetics
• Antineoplastic Agents/therapeutic use
• Drug Delivery Systems
• Extracellular Matrix/metabolism
• Humans
• Nanoparticles/administration & dosage
• Nanoparticles/therapeutic use
• Neoplasms/drug therapy
• Neoplasms/immunology
• Neoplasms/metabolism
• Tumor Microenvironment

• R43 TR000356 NCATS NIH HHS
• R44 CA107743 NCI NIH HHS
• R01 EB015253 NIBIB NIH HHS
• R43 CA107743 NCI NIH HHS
• R44 CA103133 NCI NIH HHS
• R01 GM100487 NIGMS NIH HHS
• R01EB015253 NIBIB NIH HHS
• R43TR000356 NCATS NIH HHS
• R01GM100487 NIGMS NIH HHS
• R01 CA163015 NCI NIH HHS
• R01 CA158300 NCI NIH HHS
• R43 CA162448 NCI NIH HHS
• R43CA162448 NCI NIH HHS
• R43/R44CA107743 NCI NIH HHS
• R01CA163015 NCI NIH HHS
• R43/R44CA103133 NCI NIH HHS
• R01CA158300 NCI NIH HHS

We designed a new siRNA system which turns on RNAi responding to a cancer cell-specific protease by using a peptide nucleic acid (PNA)-peptide conjugate.

Short interfering RNAs (siRNAs) are widely used as tool for gene inactivation in basic research and therapeutic applications. One of the major shortcomings of siRNA experiments are sequence-specific off-target effects. Such effects are largely unpredictable because siRNAs can affect partially complementary sequences and function like microRNAs (miRNAs), which inhibit gene expression on mRNA stability or translational levels. Here we demonstrate that novel, enzymatically generated siRNA pools—referred to as siPools—containing up to 60 accurately defined siRNAs eliminate off-target effects. This is achieved by the low concentration of each individual siRNA diluting sequence-specific off-target effects below detection limits. In fact, whole transcriptome analyses reveal that single siRNA transfections can severely affect global gene expression. However, when complex siRNA pools are transfected, almost no transcriptome alterations are observed. Taken together, we present enzymatically produced complex but accurately defined siRNA pools with potent on-target silencing but without detectable off-target effects.

• Bm5 cells
• BmCPV
• Cytoplasmic polyhedrosis virus
• RNAi
• dsRNA

• Animals
• Bombyx/virology
• Cells, Cultured
• Gene Silencing
• Luciferases
• RNA Interference
• RNA, Double-Stranded
• Reoviridae/immunology
• Nicotiana/virology
• Transfection

Gene silencing triggered by chemically synthesized small interfering RNAs (siRNAs) has become a powerful tool for deciphering gene function in many eukaryotes. However, prediction and validation of a single siRNA duplex specific to a target gene is often ineffective. RNA interference (RNAi) with synthetic siRNA suffers from lower silencing efficacy, off-target effects and is cost-intensive, especially for functional genomic studies. With the explosion of fungal genomic information, there is an increasing need to analyze gene function in a rapid manner. Therefore, studies were performed in order to investigate the efficacy of gene silencing induced by RNase III-diced-siRNAs (d-siRNA) in model filamentous fungus, Aspergillus nidulans.

Stable expression of heterologous reporter gene in A. nidulans eases the examination of a new RNAi-induction route. Hence, we have optimized Agrobacterium tumefaciens-mediated transformation (AMT) of A. nidulans for stable expression of sGFP gene. This study demonstrates that the reporter GFP gene stably introduced into A. nidulans can be effectively silenced by treatment of GFP-d-siRNAs. We have shown the down-regulation of two endogenous genes, AnrasA and AnrasB of A. nidulans by d-siRNAs. We have also elucidated the function of an uncharacterized Ras homolog, rasB gene, which was found to be involved in hyphal growth and development. Further, silencing potency of d-siRNA was higher as compared to synthetic siRNA duplex, targeting AnrasA. Silencing was shown to be sequence-specific, since expression profiles of other closely related Ras family genes in d-siRNA treated AnrasA and AnrasB silenced lines exhibited no change in gene expression.

We have developed and applied a fast, specific and efficient gene silencing approach for elucidating gene function in A. nidulans using d-siRNAs. We have also optimized an efficient AMT in A. nidulans, which is useful for stable integration of transgenes.

• Agrobacterium tumefaciens/genetics
• Aspergillus nidulans/genetics
• Aspergillus nidulans/metabolism
• Fungal Proteins/genetics
• Fungal Proteins/metabolism
• RNA Interference/physiology
• RNA, Small Interfering/genetics

Synthetic small interfering RNAs (siRNAs) are an indispensable tool to investigate gene function in eukaryotic cells^1,2 and may be used for therapeutic purposes to knockdown genes implicated in disease³. Thus far, most synthetic siRNAs have been produced by chemical synthesis. Here we present a method to produce highly potent siRNAs in E. coli. This method relies on ectopic expression of p19, a siRNA-binding protein found in a plant RNA virus^{4, 5}. When expressed in E. coli, p19 stabilizes ~21 nt siRNA-like species produced by bacterial RNase III. Transfection of mammalian cells with siRNAs, generated in bacteria expressing p19 and a hairpin RNA encoding 200 or more nucleotides of a target gene, at low nanomolar concentrations reproducibly knocks down gene expression by ~90% without immunogenicity or off-target effects. Because bacterially produced siRNAs contain multiple sequences against a target gene, they may be especially useful for suppressing polymorphic cellular or viral genes.

• Algorithms
• Amino Acid Sequence
• HIV-1/physiology
• HeLa Cells
• Humans
• MicroRNAs/genetics
• MicroRNAs/metabolism
• Molecular Sequence Data
• RNA Interference
• RNA, Viral/genetics
• Ribonuclease III/genetics
• Ribonuclease III/metabolism
• Sequence Analysis, RNA
• Virus Replication
• RNA, Small Untranslated

• Antiviral Agents/pharmacology
• Cell Line
• Gene Silencing/drug effects
• Giardia/enzymology
• Humans
• Immunity, Innate/drug effects
• Immunity, Innate/immunology
• Interferons/pharmacology
• RNA, Small Interfering/pharmacology
• Ribonuclease III/metabolism
• Simplexvirus/drug effects
• Simplexvirus/physiology
• Species Specificity
• Substrate Specificity/drug effects
• Viral Proteins/metabolism
• Virus Replication/drug effects

• Animals
• Cell Line, Tumor
• Cell Membrane Permeability
• HeLa Cells
• Humans
• Luciferases, Renilla/genetics
• Peptides/chemistry
• RNA Interference
• RNA, Small Interfering/chemistry
• RNA, Small Interfering/genetics
• RNA, Small Interfering/metabolism
• Renilla/genetics
• rev Gene Products, Human Immunodeficiency Virus/chemistry

The ability of embryonic stem (ES) cells to generate any of the around 220 cell types of the adult body has fascinated scientists ever since their discovery. The capacity to re-program fully differentiated cells into induced pluripotent stem (iPS) cells has further stimulated the interest in ES cell research. Fueled by this interest, intense research has provided new insights into the biology of ES cells in the recent past. The development of large-scale and high throughput RNAi technologies has made it possible to sample the role of every gene in maintaining ES cell identity. Here, we review the RNAi screens performed in ES cells to date and discuss the challenges associated with these large-scale experiments. Furthermore, we provide a perspective on how to streamline the molecular characterization following the initial phenotypic description utilizing bacterial artificial chromosome (BAC) transgenesis.

• Humans
• Nanomedicine/methods
• Nanoparticles
• Quality Control
• RNA Interference
• RNA, Small Interfering/chemistry
• Recombinant Proteins/chemistry
• Ribonuclease III/genetics

• HHSN261200800001C NCI NIH HHS
• HHSN261200800001E NCI NIH HHS
• R01 GM079604 NIGMS NIH HHS
• Intramural NIH HHS

• Animals
• Female
• Gene Expression Regulation
• Insect Proteins/genetics
• Insect Proteins/metabolism
• Male
• Pest Control/methods
• RNA Interference
• Reproduction
• Species Specificity
• Tephritidae/genetics
• Tephritidae/growth & development
• Tephritidae/physiology
• Transformation, Genetic

• Base Sequence
• DNA, Complementary/genetics
• DNA, Complementary/metabolism
• Databases, Nucleic Acid
• Gene Library
• Genetic Vectors
• High-Throughput Nucleotide Sequencing/methods
• Luciferases/analysis
• Molecular Sequence Data
• RNA Interference
• RNA, Small Interfering/genetics
• Retroviridae/genetics

This paper presents evidence that chromatin condensation, like nuclear envelope breakdown, is brought about through the combined effects of cyclins A2 and B1, and that cyclins B1 and B2 are largely responsible for maintenance of a spindle assembly checkpoint arrest.

Here we have used siRNAs and time-lapse epifluorescence microscopy to examine the roles of various candidate mitotic cyclins in chromatin condensation in HeLa cells. Knocking down cyclin A2 resulted in a substantial (∼7 h) delay in chromatin condensation and histone H3 phosphorylation, and expressing an siRNA-resistant form of cyclin A2 partially rescued chromatin condensation. There was no detectable delay in DNA replication in the cyclin A2 knockdowns, arguing that the delay in chromatin condensation is not secondary to a delay in S-phase completion. Cyclin A2 is required for the activation and nuclear accumulation of cyclin B1-Cdk1, raising the possibility that cyclin B1-Cdk1 mediates the effects of cyclin A2. Consistent with this possibility, we found that chromatin condensation was tightly associated temporally with the redistribution of cyclin B1 to the nucleus. Moreover, a constitutively nuclear cyclin B1 rescued chromatin condensation in cyclin A2 knockdown cells. On the other hand, knocking down cyclin B1 delayed chromatin condensation by only about one hour. Our working hypothesis is that active, nuclear cyclin B1-Cdk1 normally cooperates with cyclin A2 to bring about early mitotic events. Because cyclin A2 is present only during the early stages of mitosis, we asked whether cyclin B knockdown might have more dramatic defects on late mitotic events. Consistent with this possibility, we found that cyclin B1- and cyclin B1/B2-knockdown cells had difficulty in maintaining a mitotic arrest in the presence of nocodazole. Taken together, these data suggest that cyclin A2 helps initiate mitosis, in part through its effects on cyclin B1, and that cyclins B1 and B2 are particularly critical for the maintenance of the mitotic state.

Reactive oxygen species (ROS) are produced in growth factor signaling pathways leading to cell proliferation, but the mechanisms leading to ROS generation and the targets of ROS signals are not well understood. Using a focused siRNA screen to identify redox-related proteins required for growth factor induced cell cycle entry, we show that two ROS generating proteins, the NADPH oxidases NOX4 and DUOX2, are required for platelet-derived growth factor (PDGF) induced retinoblastoma protein (Rb) phosphorylation in normal human fibroblasts. Unexpectedly, NOX4 and DUOX2 knockdown did not inhibit the early signaling pathways leading to cyclin D1 upregulation. However, hours after growth factor stimulation, NOX4 and DUOX2 knockdown reduced ERK1 phosphorylation and increased levels of the tumor suppressor protein p53 and a cell cycle inhibitor protein p21 (Waf1/Cip1) that is transcriptionally regulated by p53. Co-knockdown of NOX4 or DUOX2 with either p53 or with p21 overcame the inhibition of Rb phosphorylation that occurred with NOX4 or DUOX2 knockdown alone. Our results argue that rather than primarily affecting growth factor receptor signaling, NOX4 and DUOX2 regulate cell cycle entry as part of a p53-dependent checkpoint for proliferation.

Chronic infection with hepatitis B virus (HBV) occurs in approximately 6% of the world's population and carriers of the virus are at risk for hepatocellular carcinoma and cirrhosis. Current treatment regimens, which include interferon-α and nucleoside/nucleotide analogs, are only partially effective and new treatment methods remain an important objective. Harnessing the RNA interference (RNAi) pathway to achieve post-transcriptional silencing of rogue genetic elements is an exciting avenue for development of novel therapeutic strategies. The specific and potent suppression of HBV gene expression and replication is an attractive option as a novel and effective approach for the treatment of chronic HBV infection. However, despite significant and rapid progress, existing RNAi technologies require further refinement before clinical applications can be realized. Here, we review current efforts aimed at improving the efficiency of anti-HBV RNAi-based delivery systems, at limiting the toxicities associated with RNAi modalities and at preventing reactivation of viral replication. We discuss the progress towards clinical implementation of anti-HBV RNAi therapies.

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.

Short RNAs derived from invading viruses with RNA genomes are important components of antiviral immunity in plants, worms and flies. The regulated generation of these short RNAs, and their engagement by the immune apparatus, is essential for inhibiting viral growth in these organisms. Mammals have the necessary protein components to generate these viral-derived short RNAs (“vsRNAs”), raising the question of whether vsRNAs in mammals are a general feature of infections with RNA viruses. Our work with Hepatitis C, Polio, Dengue, Vesicular Stomatitis, and West Nile viruses in a broad host repertoire demonstrates the generality of RNA virus-derived vsRNA production, and the ability of the cellular short RNA apparatus to engage these vsRNAs in mammalian cells. Detailed analyses of vsRNA and host-derived short RNA populations demonstrate both common and virus-specific features of the interplay between viral infection and short RNA populations. The vsRNA populations described in this work represent a novel dimension in both viral pathogenesis and host response.

RNA interference (RNAi) has become a powerful means for silencing target gene expression in mammalian cells and is envisioned to be useful in therapeutic approaches to human disease. In recent years, high-throughput, genome-wide screening of siRNA/miRNA libraries has emerged as a desirable approach. Current methods for constructing siRNA/miRNA expression vectors require the synthesis of long oligonucleotides, which is costly and suffers from mutation problems.

Here we report an ingenious method to solve traditional problems associated with construction of siRNA/miRNA expression vectors. We synthesized shorter primers (< 50 nucleotides) to generate a linear expression structure by PCR. The PCR products were directly transformed into chemically competent E. coli and converted to functional vectors in vivo via homologous recombination. The positive clones could be easily screened under UV light. Using this method we successfully constructed over 500 functional siRNA/miRNA expression vectors. Sequencing of the vectors confirmed a high accuracy rate.

This novel, convenient, low-cost and highly efficient approach may be useful for high-throughput assays of RNAi libraries.