Vigilance and validation: Keys to success in RNAi screening

Abstract

In the 12 years since the process of RNA interference (RNAi) was first discovered, great progress has been made in understanding its mechanism and exploiting its ability to silence gene expression to study gene function at a genome-wide level. Its extensive use as a screening method has yielded many published lists of genes that play novel roles in higher eukaryotes. However, the usefulness of this information is potentially limited by the occurrence of unintended off-target effects. Here we review the potential causes of off-target effects and the impact of this phenomenon in interpreting the results of high-throughput RNAi screens. In addition to targeting the intended gene product, artificial short interfering RNAs (siRNAs) can produce off-target effects by down-regulating the expression of multiple mRNAs through microRNA-like targeting of the 3' untranslated region. We examine why this phenomenon can produce high hit rates in siRNA screens and why independent validation of screening results is critical for the approach to yield new biological insights.

Figure 1

Model of mammalian RNA interference pathways. RNA molecules involved in RNAi pathways originate from artificial sources including siRNA transfection and shRNA expression from a transfected vector or endogenous sources where miRNA genes encode a pri-miRNA. A protein complex Drosha/Pasha cleaves the pri-miRNA to generate one or more pre-miRNA molecule. shRNAs and pre-miRNAs are transported into the cytoplasm by a mechanism involving Exportin5. The RNase III enzyme Dicer further cleaves shRNAs and pre-miRNAs into mature siRNAs and miRNAs, respectively. siRNA and miRNA single strands incorporate into RISC complexes and serve as templates to target mRNAs for cleavage, translation inhibition and/or mRNA decay. The fate of the targeted mRNA depends on the extent of sequence pairing.

Figure 2

Seed sequence-based interaction of siRNA guide strand with an off-target mRNA. The 7-nucleotide seed region of the siRNA guide strand (position 2–8) shows full complementarity (blue, A and B) to a target site in the 3′UTR of off-targeted mRNA. Additional siRNA 3′ sequence pairing (green, B) to the target mRNA may contribute to efficient off-targeting by the siRNA. Centered complementarity site (siRNA position 4– 14 or 5–15) to the mRNA target (red, C) can drive a RISC slicer activity-dependent cleavage of the target mRNA.