A protein sensor for siRNA asymmetry
ABSTRACT To act as guides in the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be unwound into their component strands, then assembled with proteins to form the RNA-induced silencing complex (RISC), which catalyzes target messenger RNA cleavage. Thermodynamic differences in the base-pairing stabilities of the 5' ends of the two approximately 21-nucleotide siRNA strands determine which siRNA strand is assembled into the RISC. We show that in Drosophila, the orientation of the Dicer-2/R2D2 protein heterodimer on the siRNA duplex determines which siRNA strand associates with the core RISC protein Argonaute 2. R2D2 binds the siRNA end with the greatest double-stranded character, thereby orienting the heterodimer on the siRNA duplex. Strong R2D2 binding requires a 5'-phosphate on the siRNA strand that is excluded from the RISC. Thus, R2D2 is both a protein sensor for siRNA thermodynamic asymmetry and a licensing factor for entry of authentic siRNAs into the RNAi pathway.
- SourceAvailable from: André Nicolau Aquime Gonçalves[Show abstract] [Hide abstract]
ABSTRACT: Viral RNA is a common activator of antiviral responses. In this review, we dissect the mechanism of viral RNA recognition by the small interfering RNA pathway in Drosophila melanogaster. This antiviral response in fruit flies can help understand general principles of nucleic acid recognition.Microbes and Infection 09/2014; 16(12). DOI:10.1016/j.micinf.2014.09.001 · 2.73 Impact Factor
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ABSTRACT: Neurons regulate ionic fluxes across their plasma membrane to maintain their excitable properties under varying environmental conditions. However, the mechanisms that regulate ion channels abundance remain poorly understood. Here we show that pickpocket 29 (ppk29), a gene that encodes a Drosophila degenerin/epithelial sodium channel (DEG/ENaC), regulates neuronal excitability via a protein-independent mechanism. We demonstrate that the mRNA 3'UTR of ppk29 affects neuronal firing rates and associated heat-induced seizures by acting as a natural antisense transcript (NAT) that regulates the neuronal mRNA levels of seizure (sei), the Drosophila homolog of the human Ether-à-go-go Related Gene (hERG) potassium channel. We find that the regulatory impact of ppk29 mRNA on sei is independent of the sodium channel it encodes. Thus, our studies reveal a novel mRNA dependent mechanism for the regulation of neuronal excitability that is independent of protein-coding capacity. DOI: http://dx.doi.org/10.7554/eLife.01849.001.eLife Sciences 03/2014; 3:e01849. DOI:10.7554/eLife.01849 · 8.52 Impact Factor
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ABSTRACT: RNA silencing is an established method for investigating gene function and has attracted particular interest due to the potential for generating RNA-based therapeutics. Utilising lentiviral vectors as an efficient delivery system that offers stable, long term expression in post-mitotic cells further enhances the therapeutical applicability of an RNA-based gene therapy for the CNS. In this review we provide an overview of both lentiviral vectors and RNA silencing as well as presenting design considerations for generating lentiviral vectors capable of RNA silencing. We go on to describe the current pre-clinical data regarding lentiviral vector mediated RNA silencing for CNS disorders and discuss the concerns of non-specific side-effects associated with lentiviral vectors and siRNAs and how these might be mitigated.Human Gene Therapy Methods 10/2013; DOI:10.1089/hgtb.2013.016 · 1.64 Impact Factor