Building mammalian signalling pathways with RNAi screens

Abstract | Technological advances in mammalian systems are providing new tools to identify the molecular components of signalling pathways. Foremost among these tools is the ability to knock down gene function through the use of RNA interference (RNAi). The fact that RNAi can be scaled up for use in high-throughput techniques has motivated the creation of genome-wide RNAi reagents. We are now at the brink of being able to harness the power of RNAi for large-scale functional discovery in mammalian cells.

Small interfering RNA (siRNA). A class of 19–22- nucleotide-long RNA molecules that interfere with the expression of genes by eliciting the RNAi response. siRNAs are short double-stranded RNA molecules with 2-nucleotide overhangs on either end, including a 5′ phosphate group and a 3′ hydroxyl group. They can be artificially introduced into cells to bring about the knockdown of a particular gene. Interferon response A primitive antiviral response to dsRNAs of >30 base pairs, which triggers the sequencenonspecific degradation of mRNA and the downregulation of cellular protein synthesis.

Genome sequencing has ushered in the need for new technologies for the functional annotation of human genes. This has been particularly difficult in mammalian systems because of the lack of tools to probe gene function systematically and quickly. RNA interference (RNAi) offers the cell biologist an approach to perturb gene function that can be applied in a high-throughput fashion on the cell or organism scale1,2. RNAi is a sequence-specific, post-transcriptional, gene-silencing process3–6 that is mediated by double-stranded RNA (dsRNA) molecules7. The effectors of RNAi are small interfering RNAs (siRNAs) that are processed from longer precursors by a ribo nuclease known as DICER. One strand of the siRNA functions as a template for the RNA-induced silencing complex (RISC) to pair to, and cleave, a complementary mRNA. Cleaved mRNAs are then rapidly degraded.

Long dsRNAs (400–700 base pairs) induce specific and potent gene silencing when introduced into worms, flies or plants3–6. RNAi libraries that target most genes in worms and flies have been successfully used in screens that have provided important insights into gene functions8– 12. In mammalian cells, long dsRNA triggers a nonspecific interferon response13; therefore, siRNAs14, short hairpin RNAs (shRNAs)15–19, or short hairpin RNAs in a microRNA (miRNA) context (shRNA-mirs)20–22 must be used to prevent these nonspecific effects. In the interferon response, dsRNA molecules of >30 base pairs bind to and activate the protein kinase PKR and 2′,5′-oligoadenylate synthetase, which go on to stall translation and cause mRNA degradation in a sequence-independent manner23,24. Commercial vendors and academic laboratories have now created sets of chemically synthesized siRNA reagents and have also constructed, or are in the process of constructing, large shRNA- or shRNAmir- based libraries in retroviral22,25,26, adenoviral27 and lentiviral vectors28.

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