Uncoupling of RNAi from active translation in mammalian cells.
ABSTRACT Small inhibitory RNAs (siRNAs) are produced from longer RNA duplexes by the RNAse III family member Dicer. The siRNAs function as sequence-specific guides for RNA cleavage or translational inhibition. The precise mechanism by which siRNAs direct the RNA-induced silencing complex (RISC) to find the complementary target mRNA remains a mystery. Some biochemical evidence connects RNAi with translation making attractive the hypothesis that RISC is coupled with the translational apparatus for scanning mRNAs. Such coupling would facilitate rapid alignment of the siRNA antisense with the complementary target sequence. To test this hypothesis we took advantage of a well-characterized translational switch afforded by the ferritin IRE-IRP to analyze RNAi mediated cleavage of a target mRNA in the presence and absence of translation. Our results demonstrate that neither active translation nor unidirectional scanning is required for siRNA mediated target degradation. Our findings demonstrate that nontranslated mRNAs are highly susceptible to RNAi, and blocking scanning from both the 5' and 3' ends of an mRNA does not impede RNAi. Interestingly, RNAi is about threefold more active in the absence of translation.
Full-textDOI: · Available from: Shuo Gu, May 29, 2015
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ABSTRACT: It has been noted that target sites located in the coding region or the 3'-untranslated region (3'-UTR) can be silenced to significantly different levels by the same siRNA, but little is known about at what specificity the silencing was achieved. In an exploration of positional effects on siRNA specificity by luciferase reporter system, we surprisingly discovered that siRNA had greatly elevated tolerance towards mismatches in target sites in the 3'-UTR of the mRNA compared with the same target sites cloned in the coding region. Assessment of changes in protein and mRNA levels suggested that the differential mismatch tolerance might have resulted from location-specific translational repression in the 3'-UTR. Ablation of argonaute proteins by AGO-specific siRNAs revealed that the AGO2 had major impact on siRNA silencing activity against sites in both coding region and 3'-UTR, while the silencing of nonnucleolytic AGO proteins (AGO1, AGO3 and AGO4) did not significantly affect silencing of sites in either region. This paper revealed the discovery that the specificity of an siRNA can be affected by the location of its target site.PLoS ONE 11/2012; 7(11):e49309. DOI:10.1371/journal.pone.0049309 · 3.53 Impact Factor
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ABSTRACT: Small interfering RNA (siRNA)-based therapies have great promise in the treatment of a number of prevalent pulmonary disorders including lung cancer, asthma and cystic fibrosis. However, progress in this area has been hindered due to the lack of carriers that can efficiently deliver siRNA to lung epithelial cells, and also due to challenges in developing oral inhalation (OI) formulations for the regional administration of siRNA and their carriers to the lungs. In this work we report the ability of generation four, amine-terminated poly(amidoamine) (PAMAM) dendrimer (G4NH2)-siRNA complexes (dendriplexes) to silence the enhanced green fluorescent protein (eGFP) gene on A549 lung alveolar epithelial cells stably expressing eGFP. We also report the formulation of the dendriplexes and their aerosol characteristics in propellant-based portable OI devices. The size and gene silencing ability of the dendriplexes was seen not to be a strong function of the N/P ratio. Silencing efficiencies of up to 40% are reported. Stable dispersions of the dendriplexes encapsulated in mannitol and also in a biodegradable and water-soluble co-oligomer were prepared in hydrofluoroalkane (HFA)-based pressurized metered-dose inhalers (pMDIs). Their aerosol characteristics were very favorable, and conducive to deep lung deposition, with respirable fractions of up to 77%. Importantly, siRNA formulated as dendriplexes in pMDIs was shown to keep its integrity after the particle preparation processes, and also after long-term exposures to HFA. The relevance of this study stems from the fact that this is the first work to report the formulation of inhalable siRNA with aerosol properties suitable to deep lung deposition using pMDIs devices that are the least expensive and most widely used portable inhalers. This study is relevant because, also for the first time, it shows that siRNA-G4NH2 dendriplexes can efficiently target lung alveolar epithelial A549 cells and silence genes even after siRNA has been exposed to the propellant environment.Molecular Pharmaceutics 05/2014; DOI:10.1021/mp4006358 · 4.79 Impact Factor
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ABSTRACT: The use of siRNAs to knock down gene expression can potentially be an approach to treat various diseases. To avoid siRNA toxicity the less transcriptionally active H1 pol III promoter, rather than the U6 promoter, was proposed for siRNA expression. To identify highly efficacious siRNA sequences, extensive screening is required, since current computer programs may not render ideal results. Here, we used CCR5 gene silencing as a model to investigate a rapid and efficient screening approach. We constructed a chimeric luciferase-CCR5 gene for high-throughput screening of siRNA libraries. After screening approximately 900 shRNA clones, 12 siRNA sequences were identified. Sequence analysis demonstrated that most (11 of the 12 sequences) of these siRNAs did not match those identified by available siRNA prediction algorithms. Significant inhibition of CCR5 in a T-lymphocyte cell line and primary T cells by these identified siRNAs was confirmed using the siRNA lentiviral vectors to infect these cells. The inhibition of CCR5 expression significantly protected cells from R5 HIV-1JRCSF infection. These results indicated that the high-throughput screening method allows efficient identification of siRNA sequences to inhibit the target genes at low levels of expression.PLoS ONE 05/2014; 9(5):e96445. DOI:10.1371/journal.pone.0096445 · 3.53 Impact Factor