RNAi technology and its use in studying the function of nuclear receptors and coregulators

Until just a few years ago, RNA interference (RNAi) technology was restricted to the research fields of plants, C. elegans or Drosophila. The discovery of gene silencing by in vitro synthesized double-stranded RNA (dsRNA) in mammalian cells has made the use of RNAi possible in nearly the entire life science kingdom. DNA vectors delivering small interfering RNA (siRNA) directed by polymerase III or polymerase II promoters to persistently inhibit target genes expression have extended this technology to study in vivo function of these genes. Recently, RNAi has been used as a powerful tool in the functional analysis of nuclear receptors and their coregulators. This short review will cover studies in this area

High potency silencing by single-stranded boranophosphate siRNA

In RNA interference (RNAi), double-stranded short interfering RNA (ds-siRNA) inhibits expression from complementary mRNAs. Recently, it was demonstrated that short, single-stranded antisense RNA (ss-siRNA) can also induce RNAi. While ss-siRNA may offer several advantages in both clinical and research applications, its overall poor activity compared with ds-siRNA has prevented its widespread use. In contrast to the poor gene silencing activity of native ss-siRNA, we found that the silencing activity of boranophosphate-modified ss-siRNA is comparable with that of unmodified ds-siRNA. Boranophosphate ss-siRNA has excellent maximum silencing activity and is highly effective at low concentrations. The silencing activity of boranophosphate ss-siRNA is also durable, with significant silencing up to 1 week after transfection. Thus, we have demonstrated that boranophosphate-modified ss-siRNA can silence gene expression as well as native ds-siRNA, suggesting that boranophosphate-modified ss-siRNAs should be investigated as a potential new class of therapeutic agents

A robust system for RNA interference in the chicken using a modified microRNA operon

AbstractRNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo

Estrogen inhibits GH signaling by suppressing GH-induced JAK2 phosphorylation, an effect mediated by SOCS-2

Oral estrogen administration attenuates the metabolic action of growth hormone (GH) in humans. To investigate the mechanism involved, we studied the effects of estrogen on GH signaling through Janus kinase (JAK)2 and the signal transducers and activators of transcription (STATs) in HEK293 cells stably expressing the GH receptor (293GHR), HuH7 (hepatoma) and T-47D (breast cancer) cells. 293GHR cells were transiently transfected with an estrogen receptor-α expression plasmid and luciferase reporters with binding elements for STAT3 and STAT5 or the β-casein promoter. GH stimulated the reporter activities by four- to sixfold. Cotreatment with 17β-estradiol (E2) resulted in a dose-dependent reduction in the response of all three reporters to GH to a maximum of 49-66% of control at 100 nM (P < 0.05). No reduction was seen when E2 was added 1-2 h after GH treatment. Similar inhibitory effects were observed in HuH7 and T-47D cells. E2 suppressed GH-induced JAK2 phosphorylation, an effect attenuated by actinomycin D, suggesting a requirement for gene expression. Next, we investigated the role of the suppressors of cytokine signaling (SOCS) in E2 inhibition. E2 increased the mRNA abundance of SOCS-2 but not SOCS-1 and SOCS-3 in HEK293 cells. The inhibitory effect of E2 was absent in cells lacking SOCS-2 but not in those lacking SOCS-1 and SOCS-3. In conclusion, estrogen inhibits GH signaling, an action mediated by SOCS-2. This paper provides evidence for regulatory interaction between a sex steroid and the GH/JAK/STAT pathway, in which SOCS-2 plays a central mechanistic role

siRNAs Induce Efficient RNAi Response in Bombyx mori Embryos

Short interference RNA (siRNA) is widely used in mammalian cells. In insects, however, reports concerning the suitablility of siRNA in vivo is very limited compared with that of long dsRNA, which is thought to be more effective. There is insufficient information on the essential rules of siRNA design in insects, as very few siRNAs have been tested in this context. To establish an effective method of gene silencing using siRNA in vivo in insects, we determined the effects of siRNA on seven target genes. We designed siRNAs according to a new guideline and injected them into eggs of Bombyx mori. At the mRNA level, the expression of most of these genes was successfully silenced, down to less than half the constitutive level, which in some cases led to the development of distinctive phenotypes. In addition, we observed stronger effect of siRNA both on the mRNA level and the phenotype than that of long dsRNA under comparable conditions. These results indicate that direct injection of siRNA is an effective reverse-genetics tool for the analysis of embryogenesis in vivo in insects

Solution-state structure of a fully alternately 2′-F/2′-OMe modified 42-nt dimeric siRNA construct

A high-resolution solution structure of a stable 42-nt RNA dimeric construct has been derived based on a high number of NMR observables including nuclear overhauser effects (NOEs), J-coupling constants and residual dipolar couplings (RDCs), which were all obtained with isotopically unlabeled molecules. Two 21-nt siRNA that efficiently hybridize consist of ribose units that were alternately substituted by 2′-fluoro or 2′-methoxy groups. Structure calculations utilized a set of H-F RDC values for all 21 2′-fluoro modified nucleotides under conditions of weak alignment achieved by Pf1 phages. A completely 2′-F/2′-OMe modified dimeric RNA construct adopts an antiparallel double-helical structure consisting of 19 Watson–Crick base pairs with additional 3′ UU overhangs and a 5′ phosphate group on the antisense strand. NMR data suggest that the stability of individual base pairs is not uniform throughout the construct. While most of the double helical segment exhibits well dispersed imino resonances, the last three base pairs either display uncharacteristic chemical shifts of imino protons or absence of imino resonances even at lower temperatures. Accessibility of imino protons to solvent exchange suggests a difference in stability of duplex ends, which might be of importance for incorporation of the guide siRNA strand into a RISC

RNA interference as a key to knockdown overexpressed cyclooxygenase-2 gene in tumour cells

Silencing those genes that are overexpressed in cancer and contribute to the survival and progression of tumour cells is the aim of several researches. Cyclooxygenase-2 (COX-2) is one of the most intensively studied genes since it is overexpressed in most tumours, mainly in colon cancer. The use of specific COX-2 inhibitors to treat colon cancer has generated great enthusiasm. Yet, the side effects of some inhibitors emerging during long-term treatment have caused much concern. Genes silencing by RNA interference (RNAi) has led to new directions in the field of experimental oncology. In this study, we detected sequences directed against COX-2 mRNA, that potently downregulate COX-2 gene expression and inhibit phorbol 12-myristate 13-acetate-induced angiogenesis in vitro in a specific, nontoxic manner. Moreover, we found that the insertion of a specific cassette carrying anti-COX-2 short hairpin RNA sequence into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT29) without activating any interferon response. Phenotypically, COX-2 deficient HT29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, the retroviral approach enhancing COX-2 knockdown, mediated by RNAi, proved to be an useful tool to better understand the role of COX-2 in colon cancer. Furthermore, the higher infection efficiency we observed in tumour cells, if compared to normal endothelial cells, may disclose the possibility to specifically treat tumour cells without impairing endothelial COX-2 activity

COLT-Cancer: functional genetic screening resource for essential genes in human cancer cell lines

Genome-wide pooled shRNA screens enable global identification of the genes essential for cancer cell survival and proliferation and provide a ‘functional genetic’ map of human cancer to complement genomic studies. Using a lentiviral shRNA library targeting approximately 16 000 human genes and a newly developed scoring approach, we identified essential gene profiles in more than 70 breast, pancreatic and ovarian cancer cell lines. We developed a web-accessible database system for capturing information from each step in our standardized screening pipeline and a gene-centric search tool for exploring shRNA activities within a given cell line or across multiple cell lines. The database consists of a laboratory information and management system for tracking each step of a pooled shRNA screen as well as a web interface for querying and visualization of shRNA and gene-level performance across multiple cancer cell lines. COLT-Cancer Version 1.0 is currently accessible at http://colt.ccbr.utoronto.ca/cancer