Reduced levels of Ago2 expression result in increased siRNA competition in mammalian cells

Abstract

Administration of small interfering RNAs (siRNAs) leads to degradation of specific mRNAs utilizing the cellular RNA interference (RNAi) machinery. It has been demonstrated that co-administration of siRNAs may lead to attenuation of activity of one of the siRNAs. Utilizing antisense and siRNA-mediated RNA-induced silencing complex (RISC) gene reduction we show that siRNA competition is correlated with differences in the cellular expression levels of Ago2, while levels of other RISC proteins have no effect on competition. We also show that under certain conditions siRNA competition rather than reduction of cellular RISC levels may be responsible for apparent reduction in siRNA activity. Furthermore, exploiting siRNA competition, we show that the RISC pathway loads and results in detectable cleavage of the target RNA in ∼2 h after transfection. The RISC pathway is also capable of being reloaded even in the absence of new protein synthesis. RISC reloading and subsequent induction of detectable cleavage of a new target RNA, requires about 9–12 h following the initial transfection.

Noncanonical Synthetic RNAi Inducers

“…Heterologous duplexes merged "head-to-tail" of antisense strands demonstrated reduced silencing activity [96]. Similar results were obtained for tandem siRNAs of 40-42 nt in length consisting of 21+21 and 21+23 units [101] as well as 40-nt long duplexes [100]. These results may be explained by the fact that the induction of interferon response depends on the cell type [23].…”

“…However, further experiments are required to clarify the exact mechanism of increased activity of these siRNAs. Long unmodified siRNAs up to 42 nt in length were used for silencing of gene expression in some specific cell lines without the induction of interferon response [96][97][98][99][100][101]. For example, direct fusion of two 17-and 19-bp long non-modified siRNAs resulted in efficient silencing of two target genes [96].…”

“…Recently, it has been demonstrated that branched small RNA structures may also be effective RNAi inducers. Thus, these duplexes can simultaneously inhibit two or more genes and possess improved silencing activity and intracellular delivery properties [96][97][98][99][100][101][102][103][104][105][106][107][108][109]. Different strategies are developed to form branches.…”

Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects

“…During investigation of how the individual Ago proteins contribute to siRNA on-target activity, it has been suggested that mainly the endonuclease Ago2 contributes to siRNA on-target activity (Vickers et al 2007;Diederichs et al 2008). How the individual Ago proteins contribute to siRNA off-target activity is still under discussion, but several studies have implicated that the contribution of the noncatalytic Ago proteins Ago1/3/4 to siRNA off-target activity is disproportionately high (Aleman et al 2007;Wu et al 2008;Vickers et al 2009).…”

Antisense Oligonucleotides and Therapeutical Monoclonal Antibodies as a Basement for Novel Biological Lipidlowering Drugs

“…Существует потенциальная опасность конкуренции киРНК, влияющих на целе-вую мРНК по механизму РНК-интерференции, с эндогенными микроРНК при образовании ком-плекса RISC [40]. Также было описано подавление экспрессии генов отличных от гена-мишени -так называемый "off-target" эффект, который может при-вести к нежелательным последствиям и потенциаль-ной трансформации клеток [41].…”