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Rnai and Its Applications René Ketting and Ronald Plasterk RNAi and its applications René Ketting and Ronald Plasterk Double-stranded RNA (dsRNA) molecules can be used enters the RNA-induced silencing complex (RISC), a to induce a potent silencing process, known as RNA multi-protein complex that contains an Argonaute interference (RNAi). Key players in this process are two protein through which siRNAs induce their silencing enzymes, Dicer and Argonaute, and a small RNA effect. Because RNAi is both robust and specific it has species, small interfering RNA (siRNA). The siRNA is become an important tool for gene knockdown made by the Dicer enzyme from a longer dsRNA experiments in the laboratory, and is being developed substrate. One of the strands of the duplex siRNA then for use in agriculture and in the clinic. The means of introduction • Injection of in vitro synthesized dsRNA The central component of the RISC complex is a member • Soaking in dsRNA of the Argonaute family. This protein interacts with Dicer • Feeding dsRNA-producing bacteria Injection and several other proteins, among which are dsRNA- binding proteins and RNA helicases. One of the two • Transfection of synthetic siRNA strands of the siRNA duplex, the one with the weakest Exportin 5 ′ • Transgenes that produce dsRNA molecules (hairpins) 5 end base pairing in the duplex, is selected to enter the RISC complex (this step is called strand selection). The ′ • Viruses producing dsRNA molecules 3 HO OH 3′ Argonaute protein then binds the siRNA and recruits an Drosha homologous mRNA, after which it induces mRNA AAA breakdown by introducing a nick into the mRNA. This 3′ HO Pasha cleavage reaction is carried out by the Piwi domain that Cap folds into an RNaseH-like structure. The position of the nick is always halfway along the siRNA–mRNA duplex, 3′ HO opposite the phosphodiester bond between nucleotides PAZ RNaseIII 10 and 11 of the siRNA. domain pri-miRNA In addition to mRNA breakdown, siRNAs can affect the chromatin structure of targeted genes, resulting in transcriptional inhibition. This phenomenon is mediated pre-miRNA by another complex known as RITS (RNA-induced dsRNA-binding protein transcriptional silencing). siRNAs are thought to guide chromatin modifying enzymes (e.g. histone Nucleus methyltransferases; HMTs) to specific DNA sequences, Dicer Helicase resulting in a silent chromatin structure (e.g. in methylated (m) histone H3 on lysine 9). Cytoplasm 3′ HO 3′ HO Shortly after the discovery of RNAi, it became clear that OH 3′ this pathway was used in the cell by a large family of cell-autonomous short RNA — microRNAs (miRNAs). Genome-wide RNAi The precursors of these miRNAs, the primary miRNAs (pri-miRNAs), are made in the nucleus where they are Libraries have been made that are used to silence genes in a genome- processed by Drosha into pre-miRNAs. Similar to siRNAs, wide approach for several organisms. These libraries are now used for pre-miRNAs are then processed by Dicer in the cytoplasm, Dicer-mediated cleavage gene discovery as a rapid alternative to classic genetic approaches. after which they also are bound by an Argonaute protein. Rules of the game Most methods deliver long dsRNA molecules, resulting in gene silencing miRNAs can induce silencing through mRNA cleavage, by a pool of siRNAs. Exceptions to this are the viruses that are used mainly in plants, or inhibition of translation, mainly in • Strand selection: the strand with the most loosely base-paired in humans and mice: in these cases siRNAs of only one type are being animals. The miRNA–RISC complex will specify cleavage if 5′ end in the siRNA duplex will enter the RISC complex produced, derived either from custom-made short RNA hairpins or the mRNA has sufficient complementarity to the miRNA; if the mRNA does not have sufficient complementarity to ′ hairpins that are built into larger primary miRNA transcripts. • The 5 end has to be phosphorylated siRNA miRNA be cleaved, the miRNA–RISC complex will repress mRNA • siRNAs need to have a 2-nucleotide 3′ overhang, which 3′ HO 3′ HO Injection Soaking Feeding Virus translation. mRNAs that are silenced by miRNAs are is recognized by the PAZ domain, present in Dicer and OH 3′ OH 3′ recruited to so-called processing bodies (P bodies) where Caenorhabditis elegans +++ Argonaute proteins they are either stored or degraded. • The seed sequence of miRNA (nucleotides 2–7) requires Drosophila melanogaster + perfect base pairing with target to induce silencing References Strand selection Nicotiana benthamiana + Fire, A. et al. Nature 391, 806–811 (1998) | Ma, J.-B., Ye, K. & Patel, D. J. Nature 429, 318–322 (2004) | Matzke M. A. & Birchler J. A. Nature Rev. Genet. 6, 24–35 Planaria + (2005) | McCaffrey, A. P. et al. Nature 418, 38–39 (2002) | Song, J.-J. et al. Science 305, 1434–1437 (2004) | Yuan, Y.-R. et al. Mol. Cell 19, 405–419 (2005) | Zhang, H. Mouse + et al. Cell 118, 57–68 (2004) m HP1 m HP1 m HP1 Human + René Ketting and Ronald Plasterk are at the Hubrecht Laboratory, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. H3 HMT H3 HMT H3 HMT siRNA-mediated changes in chromatin structure Image of RNaseIII structure courtesy of Lukasz Jaskiewicz and Witold Filipowicz, 3′ HO Cap 3′ HO FMI, Basel, Switzerland. Image of Argonaute structure courtesy of Yu-Ren Yuan and Dinshaw Patel, MSKCC, New York, USA. Designed by Charlotte Cartwright and Simon Fenwick. Edited by Magdalena Skipper. © 2005 Nature Publishing Group. http://www.nature.com/reviews/genetics miRNA-mediated P body Therapy and crop improvement translation prevention The specificity and robustness of RNAi have triggered AAA AAA an immense interest in using RNAi as a tool in various Argonaute 3′ HO Sigma-Aldrich http://www.sigmaaldrich.com settings. For example, RNAi is used to improve crops ? by providing resistance against parasites, and Your Source for RNA Interference Solutions modified versions of siRNAs that are directed against siRNA- miRNA- Over the course of a few short years RNA Interference has grown from an interesting disease-causing mediated mediated observation to an essential research tool in both industry and academia. The mission genes are being cleavage cleavage of Sigma-Aldrich is to make this tool available to researchers worldwide. Whether you High choose to perform your experiments using synthetic RNA or vector based shRNA we developed. Some of intensity AAA have the solution for your needs. these therapeutic 3′ HO siRNAs are already MISSION™ shRNA Libraries: As a sponsoring member of The RNAi Consortium (TRC) being tested in Sigma-Aldrich produces and distributes the lentivirus-based MISSION™ TRC shRNA Libraries consisting of 15,000 human gene targets (MISSION TRC-Hs1.0) and 15,000 clinical trials. The mouse gene targets (MISSION TRC-Mm1.0). This renewable lentiviral system allows principle of in vivo transduction of primary and non-dividing cells, as well as providing the ability to RNAi is indicated perform transient or stable silencing. The libraries are available in multiple formats — Low in the figure, where AAA bacterial glycerol stocks, purified plasmid DNA, and lentiviral particles. intensity AAA RNA luciferase in the Luciferase Reporter + Custom siRNA: Sigma-Proligo provides ready-to-use, high quality, desalted and liver is specifically reporter anti-luciferase purified custom siRNA. Our capacity ranges from microgram to multi-gram in either degradation single strand or duplex siRNA and includes a broad range of modifications. Rapid turn silenced by siRNAs. siRNA around time is achieved with our high-throughput capability. We are fully licensed and offer competitive pricing. nrg1206-poster.indd 1 8/11/05 10:13:21 am.
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