RNA interference screening demystified

Abstract

Genetic screens, where the effects of modifying gene function on cell behaviour are assessed in a systematic fashion, have for some time provided useful information to those interested in disease pathogenesis and treatment. Genetic screens exploiting the phenomenon of RNA interference (RNAi) are now becoming commonplace. This article explains the different RNAi screen formats and describes some of the applications of RNAi screening that may be pertinent to the research pathologist.

Figure 1

RNA interference. For simplicity we have shown how microRNAs (miRNAs) can mediate RNA interference in mammalian cells by causing the degradation of protein-coding transcripts. Non-protein coding miRNAs are transcribed from the genome and then processed in the nucleus into shorter RNA species bearing a hairpin structure (shRNAs); these hairpin structures, consisting of a “stem” and a “loop”, are caused by base pairing between short regions of the RNA sequence (these form the “stem”) separated by a short sequence that does not form base pairs (which forms the “loop”). shRNAs are exported from the nucleus and further processed into small RNA duplexes (siRNAs) formed from the stem of the shRNA. siRNAs are loaded into the RNA-induced silencing complex (RISC). This complex facilitates binding between one of the siRNA strands and protein-coding mRNAs that have nucleotide sequence complementary to the siRNA. Once siRNA/mRNA binding has occurred, and thus the target mRNA transcript has been recognised, a nuclease in RISC degrades the mRNA, thus ultimately reducing the amount of mRNA that is available for translation and protein production. This mechanism can be exploited experimentally to silence specific genes. Synthetic siRNAs can be delivered into cells by transfection and are readily loaded on to the RISC and mediate degradation of mRNAs with significant sequence complementarity. Viruses or plasmids containing miRNA-coding or shRNA-coding sequences can also be introduced into mammalian cells and these mimic the production of endogenous miRNA and shRNA and are processed into siRNA as before.

Figure 2

RNAi screening formats. Top panel: One gene per well screens target each gene separately in a multi-well format. Short-interfering RNAs (siRNAs), short-hairpin RNA (shRNA) plasmids or virally packaged shRNA constructs can be used to transfect or infect cells. Various readouts may be used to determine the effect of RNAi on the phenotype of interest; the measurement of cell viability is common, and luminescence-based plate readers are often used for this purpose. Alternatively, high-throughput microscopes may be used to measure cellular phenotypes in screens. Lower panel: In pooled screens, pools of shRNA-expressing vectors are introduced into cells by transfection or infection. Cells are then exposed to a selective agent such as a drug. In this case, shRNAs that cause drug resistance can be identified and quantified by amplifying shRNA sequences from genomic DNA in surviving cells. Microarray analysis is ideal for detecting shRNA sequences, as is Next Generation Sequencing. For more details, see the main text.