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RNA-induced Silencing Complex (RNA-induce + silencing_complex)
Selected AbstractsEffect of siRNA terminal mismatches on TRBP and Dicer binding and silencing efficacyFEBS JOURNAL, Issue 22 2009Hemant K. Kini To enhance silencing and avoid off-target effects, siRNAs are often designed with an intentional bias to ensure that the end of the siRNA that contains the guide strand 5, end is less stably hybridized relative to the end containing the passenger strand 5, end. One means by which this is accomplished is to introduce a terminal mismatch, typically by changing the passenger strand sequence to impair its hybridization with the guide strand 5, end. However, there are conflicting reports about the influence of terminal mismatches on the silencing efficacy of siRNAs. Here, the silencing efficiency of siRNAs with a terminal mismatch generated either by altering the guide strand (at the 5, end, nucleotide 1) or the passenger strand (nucleotide 19 from the 5, end) was examined. Subsequently, we studied the relationship between the silencing efficiency of the siRNAs and their binding to the RNA-induced silencing complex loading complex proteins HIV transactivating response RNA-binding protein and Dicer in H1299 cytoplasmic extracts. Binding of siRNA and the transactivating response RNA-binding protein was significantly reduced by terminal mismatches, which largely agrees with the reduction in eventual silencing efficacy of the siRNAs. Single terminal mismatches led to a small increase in Dicer binding, as expected, but this did not lead to an improvement in silencing activity. These results demonstrate that introduction of mismatches to control siRNA asymmetry may not always improve target silencing, and that care should be taken when designing siRNAs using this technique. [source] Optimized transfection of diced siRNA into mature primary human osteoclasts: Inhibition of cathepsin K mediated bone resorption by siRNAJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2005Christina I. Selinger Abstract Osteoclasts are large multinucleated cells responsible for bone resorption. Bone resorption is dependent on the liberation of calcium by acid and protease destruction of the bone matrix by proteinases. The key proteinase produced by the osteoclast is cathepsin K. Targeted knock-down of cathepsin K was performed using small inhibitory RNA (siRNA). siRNA is a method that introduces short double-stranded RNA molecules that instruct the RNA-induced silencing complex (RISC) to degrade mRNA species complementary to the siRNA. Transfection of siRNA by lipid cations allows for short-term inhibition of expression of the targeted gene. We show that transfection of primary human osteoclasts with siRNA to cathepsin K reduces expression by ,60% and significantly inhibits bone resorption with a reduction of both resorption pit numbers (P,=,0.018) and resorbed area (P,=,0.013). We also show that FuGENE 6 is an effective lipid transfection reagent with which to transfect primary human osteoclasts, that does not produce off-target effects. © 2005 Wiley-Liss, Inc. [source] Polerovirus protein P0 prevents the assembly of small RNA-containing RISC complexes and leads to degradation of ARGONAUTE1THE PLANT JOURNAL, Issue 3 2010Tibor Csorba Summary RNA silencing plays an important role in plants in defence against viruses. To overcome this defence, plant viruses encode suppressors of RNA silencing. The most common mode of silencing suppression is sequestration of double-stranded RNAs involved in the antiviral silencing pathways. Viral suppressors can also overcome silencing responses through protein,protein interaction. The poleroviral P0 silencing suppressor protein targets ARGONAUTE (AGO) proteins for degradation. AGO proteins are the core component of the RNA-induced silencing complex (RISC). We found that P0 does not interfere with the slicer activity of pre-programmed siRNA/miRNA containing AGO1, but prevents de novo formation of siRNA/miRNA containing AGO1. We show that the AGO1 protein is part of a high-molecular-weight complex, suggesting the existence of a multi-protein RISC in plants. We propose that P0 prevents RISC assembly by interacting with one of its protein components, thus inhibiting formation of siRNA/miRNA,RISC, and ultimately leading to AGO1 degradation. Our findings also suggest that siRNAs enhance the stability of co-expressed AGO1 in both the presence and absence of P0. [source] Immunohistochemical analysis of RNA-induced silencing complex-related proteins AGO2 and TNRC6A in prostate and esophageal cancersAPMIS, Issue 4 2010NAM JIN YOO Yoo NJ, Hur SY, Kim MS, Lee JY, Lee SH. Immunohistochemical analysis of RNA-induced silencing complex-related proteins AGO2 and TNRC6A in prostate and esophageal cancers. APMIS 2010; 118: 271,6. Evidence exists that microRNA (miRNA), which regulates gene expression, is frequently deregulated in cancers. A mature miRNA directs a RNA-induced silencing complex (RISC) to its target messenger RNA, and causes inhibition of gene transcription. Ago proteins and TNRC proteins are main components of the RISC and participate in miRNA-induced gene silencing. However, expression status of Ago and TNRC proteins has not yet been studied in human cancer tissues. In this study, we attempted to explore whether expressions of Ago2 and TNRC6A are altered in prostate carcinomas (PCA) and esophageal squamous cell carcinomas (ESCC). We analyzed the expression of Ago2 and TNRC6A in 107 PCA and 58 ESCC tissues by immunohistochemistry using a tissue microarray (TMA) method. In the prostate, Ago2 was not expressed in normal glandular cells, while it was expressed in 50.0% of prostate intraepithelial neoplasia (PIN) and 57.0% of the PCA. TNRC6A was not expressed in normal prostate cells, while it was expressed in 55.0% of the PIN and 63.6% of the PCA in cytoplasm and nucleus. In the esophagus, neither Ago2 nor TNRC6A was expressed in normal squamous cells, while Ago2 and TNRC6A were expressed in 58.6% and 62.1% of the ESCC, respectively. However, neither the expression of Ago2 or TNRC6A was associated with pathologic characteristics of the cancers, including age, sex, Gleason score (PCA) and stage. The increased expressions of Ago2 and TNRC6A in both PCA and ESCC compared with their normal cells suggested that over-expression of these proteins may be related to miRNA functions and might play a role in tumorigenesis of PCA and ESCC. [source] Viral suppression of RNA silencing: 2b wins the Golden Fleece by defeating ArgonauteBIOESSAYS, Issue 4 2007Virginia Ruiz-Ferrer In plants, virus-derived double-stranded RNA is processed into small interfering (si)RNAs by RNAse III-type enzymes. siRNAs are believed to guide an RNA-induced silencing complex (RISC) to promote sequence-specific degradation (or ,slicing') of homologous viral transcripts. This process, called RNA silencing, likely involves Argonaute (AGO) proteins that are known components of plant and animal RISCs. Plant viruses commonly counteract the silencing immune response by producing suppressor proteins, but the molecular basis of their action has remained largely unclear. A recent study by Zhang and colleagues1 now shows that the 2b suppressor of Cucumber mosaic virus directly interacts with Arabidopsis AGO1 and inhibits its slicing activity, suggesting that AGO1 might be a component of the elusive plant antiviral RISC. BioEssays 29:319,323, 2007. © 2007 Wiley Periodicals, Inc. [source] Exploring Chemical Modifications for siRNA Therapeutics: A Structural and Functional OutlookCHEMMEDCHEM, Issue 3 2010Siddharth Shukla Abstract RNA interference (RNAi) is a post-transcriptional gene silencing mechanism induced by small interfering RNAs (siRNAs) and micro-RNAs (miRNAs), and has proved to be one of the most important scientific discoveries made in the last century. The robustness of RNAi has opened up new avenues in the development of siRNAs as therapeutic agents against various diseases including cancer and HIV. However, there had remained a lack of a clear mechanistic understanding of messenger RNA (mRNA) cleavage mediated by Argonaute2 of the RNA-induced silencing complex (RISC), due to inadequate structural data. The X-ray crystal structures of the Argonaute (Ago),DNA,RNA complexes reported recently have proven to be a breakthrough in this field, and the structural details can provide guidelines for the design of the next generation of siRNA therapeutics. To harness siRNAs as therapeutic agents, the prudent use of various chemical modifications is warranted to enhance nuclease resistance, prevent immune activation, decrease off-target effects, and to improve pharmacokinetic and pharmacodynamic properties. The focus of this review is to interpret the tolerance of various chemical modifications employed in siRNAs toward RNAi by taking into account the crystal structures and biochemical studies of Ago,RNA complexes. Moreover, the challenges and recent progress in imparting druglike properties to siRNAs along with their delivery strategies are discussed. [source] | |||||||||||||