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

Transcription of Muscle Actin Genes by a Nuclear Form of Mitochondrial RNA Polymerase

Actins are the major constituent of the cytoskeleton. In this report we present several lines of evidence that muscle actin genes are transcribed by nuclear isoform of mitochondrial RNA polymerase (spRNAP-IV) whereas the non-muscle actin genes are transcribed by the conventional RNA polymerase II (PolII). We show that mRNA level of muscle actin genes are resistant to PolII inhibitors α-amanitin and triptolide as well as insensitive to knockdown of PolII but not to knockdown of spRNAP-IV, in contrast to non-muscle actin genes in several cell lines. Similar results are obtained from nuclear run-on experiments. Reporter assay using muscle actin or PolII gene promoters also demonstrate the differential sensitivity to PolII inhibitors. Finally, chromatin-immunoprecipitation experiment was used to demonstrate that spRNAP-IV is associated with promoter of muscle actin genes but not with that of non-muscle gene and knockdown of spRNAP-IV depleted this polymerase from muscle actin genes. In summary, these experiments indicate that the two types of actin genes are transcribed by different transcription machinery. We also found that POLRMT gene is transcribed by spRNAP-IV, and actin genes are sensitive to oligomycin, suggesting a transcription coupling between mitochondria and nucleus

Identification of an alternative triglyceride biosynthesis pathway

Triacylglycerols (TAGs) are the main source of stored energy in the body, providing an important substrate pool for mitochondrial beta-oxidation. Imbalances in the amount of TAGs are associated with obesity, cardiac disease and various other pathologies 1,2. In humans, TAGs are synthesized from excess, coenzyme A-conjugated fatty acids by diacylglycerol O-acyltransferases (DGAT1 and DGAT2) 3. In other organisms, this activity is complemented by additional enzymes 4, but whether such alternative pathways exist in humans remains unknown. Here we disrupt the DGAT pathway in haploid human cells and use iterative genetics to reveal an unrelated TAG-synthesizing system composed of a protein we called DIESL (also known as TMEM68, an acyltransferase of previously unknown function) and its regulator TMX1. Mechanistically, TMX1 binds to and controls DIESL at the endoplasmic reticulum, and loss of TMX1 leads to the unconstrained formation of DIESL-dependent lipid droplets. DIESL is an autonomous TAG synthase, and expression of human DIESL in Escherichia coli endows this organism with the ability to synthesize TAG. Although both DIESL and the DGATs function as diacylglycerol acyltransferases, they contribute to the cellular TAG pool under specific conditions. Functionally, DIESL synthesizes TAG at the expense of membrane phospholipids and maintains mitochondrial function during periods of extracellular lipid starvation. In mice, DIESL deficiency impedes rapid postnatal growth and affects energy homeostasis during changes in nutrient availability. We have therefore identified an alternative TAG biosynthetic pathway driven by DIESL under potent control by TMX1. </p

Haploid genetic screens identify SPRING/C12ORF49 as a determinant of SREBP signaling and cholesterol metabolism

The sterol-regulatory element binding proteins (SREBP) are central transcriptional regulators of lipid metabolism. Using haploid genetic screens we identify the SREBPRegulating Gene (SPRING/C12ORF49) as a determinant of the SREBP pathway. SPRING is a glycosylated Golgi-resident membrane protein and its ablation in Hap1 cells, Hepa1-6 hepatoma cells, and primary murine hepatocytes reduces SREBP signaling. In mice, Spring deletion is embryonic lethal yet silencing of hepatic Spring expression also attenuates the SREBP response. Mechanistically, attenuated SREBP signaling in SPRING(KO) cells results from reduced SREBP cleavage-activating protein (SCAP) and its mislocalization to the Golgi irrespective of the cellular sterol status. Consistent with limited functional SCAP in SPRING(KO) cells, reintroducing SCAP restores SREBP-dependent signaling and function. Moreover, in line with the role of SREBP in tumor growth, a wide range of tumor cell lines display dependency on SPRING expression. In conclusion, we identify SPRING as a previously unrecognized modulator of SREBP signaling

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

The Translation Initiation Factor 3f (eIF3f) Exhibits a Deubiquitinase Activity Regulating Notch Activation

The translation initiation factor complex eIF3f has an intrinsic deubiquitinase activity and regulates the Notch signaling pathway

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

Circular Single-Stranded Synthetic DNA Delivery Vectors for MicroRNA

Single-stranded (ss) circular oligodeoxynucleotides were previously found to undergo rolling circle transcription (RCT) by phage and bacterial RNA polymerases (RNAPs) into tandemly repetitive RNA multimers. Here, we redesign them to encode minimal primary miRNA mimics, with the long term aim of intracellular transcription followed by RNA processing and maturation via endogenous pathways. We describe an improved method for circularizing ss synthetic DNA for RCT by using a recently described thermostable RNA ligase, which does not require a splint oligonucleotide to juxtapose the ligating ends. In vitro transcription of four templates demonstrates that the secondary structure inherent in miRNA-encoding vectors does not impair their RCT by RNAPs previously shown to carry out RCT. A typical primary-miRNA rolling circle transcript was accurately processed by a human Drosha immunoprecipitate, indicating that if human RNAPs prove to be capable of RCT, the resulting transcripts should enter the endogenous miRNA processing pathway in human cells. Circular oligonucleotides are therefore candidate vectors for small RNA delivery in human cells, which express RNAPs related to those tested here

Targeting of human interleukin-12B by small hairpin RNAs in xenografted psoriatic skin

<p>Abstract</p> <p>Background</p> <p>Psoriasis is a chronic inflammatory skin disorder that shows as erythematous and scaly lesions. The pathogenesis of psoriasis is driven by a dysregulation of the immune system which leads to an altered cytokine production. Proinflammatory cytokines that are up-regulated in psoriasis include tumor necrosis factor alpha (TNFα), interleukin-12 (IL-12), and IL-23 for which monoclonal antibodies have already been approved for clinical use. We have previously documented the therapeutic applicability of targeting TNFα mRNA for RNA interference-mediated down-regulation by anti-TNFα small hairpin RNAs (shRNAs) delivered by lentiviral vectors to xenografted psoriatic skin. The present report aims at targeting mRNA encoding the shared p40 subunit (IL-12B) of IL-12 and IL-23 by cellular transduction with lentiviral vectors encoding anti-IL12B shRNAs.</p> <p>Methods</p> <p>Effective anti-IL12B shRNAs are identified among a panel of shRNAs by potency measurements in cultured cells. The efficiency and persistency of lentiviral gene delivery to xenografted human skin are investigated by bioluminescence analysis of skin treated with lentiviral vectors encoding the luciferase gene. shRNA-expressing lentiviral vectors are intradermally injected in xenografted psoriatic skin and the effects of the treatment evaluated by clinical psoriasis scoring, by measurements of epidermal thickness, and IL-12B mRNA levels.</p> <p>Results</p> <p>Potent and persistent transgene expression following a single intradermal injection of lentiviral vectors in xenografted human skin is reported. Stable IL-12B mRNA knockdown and reduced epidermal thickness are achieved three weeks after treatment of xenografted psoriatic skin with lentivirus-encoded anti-IL12B shRNAs. These findings mimick the results obtained with anti-TNFα shRNAs but, in contrast to anti-TNFα treatment, anti-IL12B shRNAs do not ameliorate the psoriatic phenotype as evaluated by semi-quantitative clinical scoring and by immunohistological examination.</p> <p>Conclusions</p> <p>Our studies consolidate the properties of lentiviral vectors as a tool for potent gene delivery and for evaluation of mRNA targets for anti-inflammatory therapy. However, in contrast to local anti-TNFα treatment, the therapeutic potential of targeting IL-12B at the RNA level in psoriasis is questioned.</p