Comparison of siRNA-induced off-target RNA and protein effects

Abstract

The downregulation of many mRNAs has been observed through bioinformatic analysis of microarray results following transfection of short interfering RNAs (siRNAs). Many of these mRNA changes are due to the interaction of the siRNA guide strand with partially complementary sites and thus are considered ''off-target'' effects. To examine the mRNA:siRNA interactions important for off-target effects, we generated a panel of mRNA:siRNA combinations containing single and double mismatches, bulges, and noncanonical base-pairing interactions in the 9th, 10th, and 11th positions of two siRNA binding sites located in the 39 UTR of an integrated reporter gene. Approximately half of the mRNA:siRNA combinations containing mismatches in positions 9-11 result in a twofold or more mRNA decrease with varying degrees of protein knockdown. However, mRNA and protein analysis of the various mRNA:siRNA combinations reveals instances in which mRNA and protein levels do not correlate. Analysis of the resulting degradation products recovered from an imperfectly complementary siRNA interaction with an endogenous gene reveals a small fraction of products that map to the canonical siRNA cleavage site. Furthermore, downregulation of ARGONAUTE 2 (AGO2), the only AGO family protein known to catalyze canonical siRNAmediated cleavage, did not significantly affect the degree of mRNA knockdown observed for one of the stably expressed reporters after transfection of an imperfectly complementary siRNA. These results indicate that although some degree of canonical siRNA cleavage can take place between a siRNA and an off-target transcript, most off-target mRNA reductions are likely attributable to AGO2-independent degradation processes.

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

“…In fact, offtargeting causes a significant bias in high-throughput RNAi screens Off-targeting is concentration dependent, it could be attributed to both siRNA strands, and a portion of off-targeting appears to be caused by partial complementarity between a siRNA and its target, reminiscent of the 5' seed regions of miRNAs Birmingham et al, 2006;Jackson et al, 2006b;Aleman et al, 2007;Qiu et al, 2007). In some cases of off-targeting, no correlation between predicted and actual off-target effects was reported (Hanning et al, 2013).…”

Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits

“…Long siRNAs (Ͼ29 nt) and shRNAs are prone to activate off-target gene expression (15). For patient safety, shRNA will need to be able to be switched off, currently a hurdle in viral delivery systems.…”

MicroRNAs repress translation of m7Gppp-capped target mRNAs in vitro by inhibiting initiation and promoting deadenylation

“…In contrast, base-pairing between the mRNA target site and the 3Ј-proximal part of the miRNA is much less critical, though good pairing here can rescue repression if the base-pairing of the seed is marginally suboptimal. In addition, recent publications suggest that the exact configuration of the mismatches, the mRNA sequences flanking the target sites, and the distance separating target sites may influence the efficiency of repression and/ or the preferred mechanism of repression (Aleman et al 2007;Grimson et al 2007).…”

“…This increased degradation is via the normal pathway of deadenylation followed by decapping and then degradation of the body of the mRNA, not via an initial endonucleolytic cleavage such as occurs in RNA interference with siRNAs that have perfect complementarity to the target mRNA (Behm-Ansmant et al 2006). At present it is unclear why the quantitative significance of this miRNA-mediated degradation should differ between different target mRNA/miRNA pairs: whether it is an intrinsic feature of the mRNA sequence or of the particular miRNA or, as has recently been suggested (Aleman et al 2007), the exact configuration of the mismatches in the miRNA-mRNA interaction.…”