Specific inhibition of diverse pathogens in human cells by synthetic microRNA-like oligonucleotides inferred from RNAi screens

Abstract

Systematic genetic perturbation screening in human cells remains technically challenging. Typically, large libraries of chemically synthesized siRNA oligonucleotides are used, each designed to degrade a specific cellular mRNA via the RNA interference (RNAi) mechanism. Here, we report on data from three genome-wide siRNA screens, conducted to uncover host factors required for infection of human cells by two bacterial and one viral pathogen. We find that the majority of phenotypic effects of siRNAs are unrelated to the intended "on-target" mechanism, defined by full complementarity of the 21-nt siRNA sequence to a target mRNA. Instead, phenotypes are largely dictated by "off-target" effects resulting from partial complementarity of siRNAs to multiple mRNAs via the "seed" region (i.e., nucleotides 2-8), reminiscent of the way specificity is determined for endogenous microRNAs. Quantitative analysis enabled the prediction of seeds that strongly and specifically block infection, independent of the intended ontarget effect. This prediction was confirmed experimentally by designing oligos that do not have any on-target sequence match at all, yet can strongly reproduce the predicted phenotypes. Our results suggest that published RNAi screens have primarily, and unintentionally, screened the sequence space of microRNA seeds instead of the intended on-target space of protein-coding genes. This helps to explain why previously published RNAi screens have exhibited relatively little overlap. Our analysis suggests a possible way of identifying "seed reagents" for controlling phenotypes of interest and establishes a general strategy for extracting valuable untapped information from past and future RNAi screens.high-throughput RNAi screening | antimicrobials

Genome-Wide Small Interfering RNA Screens Reveal VAMP3 as a Novel Host Factor Required for Uukuniemi Virus Late Penetration

“…When siRNA screens have been performed in different laboratories to identify host factors against a virus, the genes in published hit lists have shown poor overlap, suggesting serious inherent problems in the approach. This is in part explained by different screening procedures and analysis, as well as by siRNA off-target effects (31)(32)(33). When our analysis was limited to the 13,000 transcribed genes, the number of candidates was reduced to about 70% of the original lists independently of the Qiagen or Dharmacon screens.…”

High-Throughput Small Interfering RNA Screening Identifies Phosphatidylinositol 3-Kinase Class II Alpha as Important for Production of Human Cytomegalovirus Virions

“…False-positive results can be found in siRNA screens due to off-target binding of siRNAs. Off-target siRNA binding is most likely to occur in the 3= untranslated region (3= UTR) of mRNA (24,25), where siRNAs exhibit microRNA-like properties upon binding of a limited number of bases, akin to a microRNA seed region, to mRNA (24)(25)(26). Indeed, it has been proposed that the data returned from some screens are the result of unintentional screening of partial seed sequence matches and not on-target binding of siRNA (26).…”

“…Off-target siRNA binding is most likely to occur in the 3= untranslated region (3= UTR) of mRNA (24,25), where siRNAs exhibit microRNA-like properties upon binding of a limited number of bases, akin to a microRNA seed region, to mRNA (24)(25)(26). Indeed, it has been proposed that the data returned from some screens are the result of unintentional screening of partial seed sequence matches and not on-target binding of siRNA (26). Therefore, using genome-wide enrichment of seed sequence (GESS) matches (17,18), we investigated if the siRNAs screened in this study (all 4 siRNAs that constitute the pools of siRNAs used) were enriched with seed sequences that could bind either human 3= UTRs or full human mRNA transcripts.…”

Properties of short double-stranded RNAs carrying randomized base pairs: toward better controls for RNAi experiments

“…Furthermore, comparing the performance of AS/RND4 with that of siRND4 revealed that the former is ∼16-fold more potent, consistent with their presumed siRNA-like and miRNA-like mechanisms, respectively. In a post-analysis of three genome-wide siRNA screens conducted to uncover genes involved in bacterial/viral infections (Franceschini et al 2014), investigators concluded that the majority of the phenotypic effects were not derived from the full siRNA sequence but rather from the siRNA seed regions. To assess the effects of an rnd-siRNA with randomized positions in the seed on a whole-transcriptome scale, we transfected HeLa cells with siRND2 at 40 nM and purified total RNA 72 h post-transfection.…”

“…In addition to Ago2-mediated sequence-dependent OTEs, additional sequence-dependent OTEs can derive from RISC activation of the passenger strand (Clark et al 2008), immunostimulation (Sledz et al 2003;Hornung et al 2005;Judge et al 2005), and RISC-mediated silencing of mRNAs in a microRNA (miRNA)-like fashion (Doench et al 2003;Saxena et al 2003;Birmingham et al 2006;Jackson et al 2006;Aleman et al 2007;Vickers et al 2009;Shin et al 2010;Marine et al 2012). Indeed, a recent study declared that most of the phenotypic effects in three independent genome-wide siRNA screens stemmed from miRNA-like OTEs rather than from on-target silencing (Franceschini et al 2014). Sequence-independent OTEs are also known and derive from saturation of RNAi machinery components (Jackson and Linsley 2010) and cell toxicity leading to cell death/growth inhibition.…”