Controlled expression of functional miR-122 with a ligand inducible expression system

<p>Abstract</p> <p>Background</p> <p>To study the biological function of miRNAs, and to achieve sustained or conditional gene silencing with siRNAs, systems that allow controlled expression of these small RNAs are desirable. Methods for cell delivery of siRNAs include transient transfection of synthetic siRNAs and expression of siRNAs in the form of short hairpins using constitutive RNA polymerase III promoters. Systems employing constitutive RNA polymerase II promoters have been used to express miRNAs. However, for many experimental systems these methods do not offer sufficient control over expression.</p> <p>Results</p> <p>We present an inducible mammalian expression system that allows for the conditional expression of short hairpin RNAs that are processed <it>in vivo </it>to generate miRNAs or siRNAs. Using modified nuclear receptors in a two hybrid format and a synthetic ligand, the Rheoswitch system allows rapid and reversible induction of mRNA expression. We evaluated the system's properties using miR-122 as a model miRNA. A short hairpin encoding miR-122 cloned into the expression vector was correctly processed to yield mature miRNA upon induction with ligand and the amount of miRNA produced was commensurate with the concentration of ligand. miR-122 produced in this way was capable of silencing both endogenous target genes and appropriately designed reporter genes. Stable cell lines were obtained, resulting in heritable, consistent and reversible expression of miR-122, a significant advantage over transient transfection. Based on these results, obtained with a microRNA we adapted the method to produce a desired siRNA by designing short hairpins that can be accurately and efficiently processed.</p> <p>Conclusion</p> <p>We established an Inducible expression system with a miR-122 backbone that can be used for functional studies of miRNAs and their targets, in heterologous cells that do not normally express the miRNA. Additionally we demonstrate the feasibility of using the miR-122 backbone to express shRNA with a desired siRNA guide strand for inducible RNAi silencing.</p

EcR-B1 and Usp nuclear hormone receptors regulate expression of the VM32E eggshell gene during Drosophila oogenesis

AbstractEcdysone signaling plays key roles in Drosophila oogenesis, as its activity is required at multiple steps during egg chamber maturation. Recently, its involvement has been reported on eggshell production by controlling chorion gene transcription and amplification. Here, we present evidence that ecdysone signaling also controls the expression of the eggshell gene VM32E, whose product is a component of vitelline membrane and endochorion layers. Specifically blocking the function of the different Ecdysone receptor (EcR) isoforms we demonstrate that EcR-B1 is responsible for ecdysone-mediated VM32E transcriptional regulation. Moreover, we show that the EcR partner Ultraspiracle (Usp) is also necessary for VM32E expression. By analyzing the activity of specific VM32E regulatory regions in usp2 clones we identify the promoter region mediating ecdysone-dependent VM32E expression. By in vitro binding assay and site-directed mutagenesis we demonstrate that this region contains a Usp binding site necessary for VM32E regulation.Our results further support the crucial role of ecdysone signaling in controlling transcription of eggshell structural genes and suggest that the heterodimeric complex EcR-B1/Usp mediates the ecdysone-dependent VM32E transcriptional activation in the main body follicle cells

Identification of Ecdysone Hormone Receptor Agonists as a Therapeutic Approach for Treating Filarial Infections

Background A homologue of the ecdysone receptor has previously been identified in human filarial parasites. As the ecdysone receptor is not found in vertebrates, it and the regulatory pathways it controls represent attractive potential chemotherapeutic targets. Methodology/ Principal Findings Administration of 20-hydroxyecdysone to gerbils infected with B. malayi infective larvae disrupted their development to adult stage parasites. A stable mammalian cell line was created incorporating the B. malayi ecdysone receptor ligand-binding domain, its heterodimer partner and a secreted luciferase reporter in HEK293 cells. This was employed to screen a series of ecdysone agonist, identifying seven agonists active at sub-micromolar concentrations. A B. malayi ecdysone receptor ligand-binding domain was developed and used to study the ligand-receptor interactions of these agonists. An excellent correlation between the virtual screening results and the screening assay was observed. Based on both of these approaches, steroidal ecdysone agonists and the diacylhydrazine family of compounds were identified as a fruitful source of potential receptor agonists. In further confirmation of the modeling and screening results, Ponasterone A and Muristerone A, two compounds predicted to be strong ecdysone agonists stimulated expulsion of microfilaria and immature stages from adult parasites. Conclusions The studies validate the potential of the B. malayi ecdysone receptor as a drug target and provide a means to rapidly evaluate compounds for development of a new class of drugs against the human filarial parasites

Substrate Cooperativity in Marine Luciferases

Marine luciferases are increasingly used as reporters to study gene regulation. These luciferases have utility in bioluminescent assay development, although little has been reported on their catalytic properties in response to substrate concentration. Here, we report that the two marine luciferases from the copepods, Gaussia princeps (GLuc) and Metridia longa (MLuc) were found, surprisingly, to produce light in a cooperative manner with respect to their luciferin substrate concentration; as the substrate concentration was decreased 10 fold the rate of light production decreased 1000 fold. This positive cooperative effect is likely a result of allostery between the two proposed catalytic domains found in Gaussia and Metridia. In contrast, the marine luciferases from Renilla reniformis (RLuc) and Cypridina noctiluca (CLuc) demonstrate a linear relationship between the concentration of their respective luciferin and the rate of light produced. The consequences of these enzyme responses are discussed

Nanopore ReCappable sequencing maps SARS-CoV-2 5′ capping sites and provides new insights into the structure of sgRNAs

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5 ' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs

Molecular Evidence for a Functional Ecdysone Signaling System in Brugia malayi

Filarial parasites such as Brugia malayi and Onchocerca volvulus are the causative agents of the tropical diseases lymphatic filariasis and onchocerciasis, which infect 150 million people, mainly in Africa and Southeast Asia. Filarial nematodes have a complex life cycle that involves transmission and development within both mammalian and insect hosts. The successful completion of the life cycle includes four molts, two of which are triggered upon transmission from one host to the other, human and mosquito, respectively. Elucidation of the molecular mechanisms involved in the molting processes in filarial nematodes may yield a new set of targets for drug intervention. In insects and other arthropods molting transitions are regulated by the steroid hormone ecdysone that interacts with a specialized hormone receptor composed of two different proteins belonging to the family of nuclear receptors. We have cloned from B. malayi two members of the nuclear receptor family that show many sequence and biochemical properties consistent with the ecdysone receptor of insects. This finding represents the first report of a functional ecdysone receptor homolog in nematodes. We have also established a transgenic hormone induction assay in B. malayi that can be used to discover ecdysone responsive genes and potentially lead to screening assays for active compounds for pharmaceutical development

Selection of a Mimotope Peptide of S-adenosyl-l-homocysteine and Its Application in Immunoassays

A competitive immunoassay for S-adenosyl-l-homocysteine (SAH) has been used in the clinical test for homocysteine via an enzymatic conversion reaction. Since S-adenosyl-l-homocysteine is a relatively unstable compound, we have used peptide library phage display to select a new mimotope peptide that interacts with the anti-SAH antibody. By immobilizing the synthetic peptide on solid phase as a competitive surrogate for SAH, we demonstrate its utility in a competitive ELISA assay. The linear range of the assay for SAH was 0.4–6.4 µM, in good correlation to the conventional assay using an SAH-conjugated plate. Our results show that the mimotope peptide has potential to substitute for SAH in immunoassays

Selection of a Mimotope Peptide of S-adenosyl-l-homocysteine and Its Application in Immunoassays

A competitive immunoassay for S-adenosyl-l-homocysteine (SAH) has been used in the clinical test for homocysteine via an enzymatic conversion reaction. Since S-adenosyl-l-homocysteine is a relatively unstable compound, we have used peptide library phage display to select a new mimotope peptide that interacts with the anti-SAH antibody. By immobilizing the synthetic peptide on solid phase as a competitive surrogate for SAH, we demonstrate its utility in a competitive ELISA assay. The linear range of the assay for SAH was 0.4–6.4 µM, in good correlation to the conventional assay using an SAH-conjugated plate. Our results show that the mimotope peptide has potential to substitute for SAH in immunoassays