RNA interferenceSo that I don’t get myself into any trouble, most of the followingtext is verbatim from the Ambion website, which nicely summarizesa lot of what I will talk about in class. I’ve edited it a bit and pastedin some figures to help, but I’m not keeping tract of what thewebsite had and what I’ve inserted. If you want to see the originalAmbion text, go tohttp://www.ambion.com/techlib/hottopics/rnai/ .The first experimentsPost-transcriptional gene silencing (PTGS), which was initiallyconsidered a bizarre phenomenon limited to petunias and a fewother plant species, is now one of the hottest topics in molecularbiology (1). In the last few years, it has become clear that PTGSoccurs in both plants and animals and has roles in viral defense andtransposon silencing mechanisms. Perhaps most exciting, however,is the emerging use of PTGS and, in particular, RNA interference(RNAi) — PTGS initiated by the introduction of double-stranded RNA(dsRNA) — as a tool to knock out expression of specific genes in avariety of organisms (reviewed in 1-3).How was RNAi discovered? How does it work? Perhaps moreimportantly, how can it be harnessed for functional genomicsexperiments? This article will briefly answer these questions andprovide you with resources to find in depth information on PTGSand RNAi research.Figure 1. The attempt to overexpress chalone synthetase, an enzyme that producesanthocyanin pigment, resulted not in a darker petunia flower, but instead a loss offlower pigment.More than a decade ago, a surprising observation was made inpetunias. While trying to deepen the purple color of these flowers,Rich Jorgensen and colleagues introduced a pigment-producinggene under the control of a powerful promoter. Instead of theexpected deep purple color, many of the flowers appearedvariegated or even white. Jorgensen named the observedphenomenon "cosuppression", since the expression of both theintroduced gene and the homologous endogenous gene wassuppressed (1-5).First thought to be a quirk of petunias, cosuppression has sincebeen found to occur in many species of plants. It has also beenobserved in fungi, and has been particularly well characterized inNeurospora crassa, where it is known as "quelling" (1-3).But what causes this gene silencing effect? Although transgene-induced silencing in some plants appears to involve gene-specificmethylation (transcriptional gene silencing, or TGS), in otherssilencing occurs at the post-transcriptional level (post-transcriptional gene silencing, or PTGS). Nuclear run-onexperiments in the latter case show that the homologous transcriptis made, but that it is rapidly degraded in the cytoplasm and doesnot accumulate (1, 3, 6).Introduction of transgenes can trigger PTGS, however silencing canalso be induced by the introduction of certain viruses (2, 3). Oncetriggered, PTGS is mediated by a diffusible, trans-acting molecule.This was first demonstrated in Neurospora, when Cogoni andcolleagues showed that gene silencing could be transferredbetween nuclei in heterokaryotic strains (1, 7). It was laterconfirmed in plants when Palauqui and colleagues induced PTGS ina host plant by grafting a silenced, transgene-containing sourceplant to an unsilenced host (8). From work done in nematodes andflies, we now know that the trans-acting factor responsible for PTGSin plants is dsRNA (1-3).RNAi Is Discovered in NematodesThe first evidence that dsRNA could lead to gene silencing camefrom work in the nematode Caenorhabditis elegans. Seven yearsago, researchers Guo and Kemphues were attempting to useantisense RNA to shut down expression of the par-1 gene in orderto assess its function. As expected, injection of the antisense RNAdisrupted expression of par-1, but quizzically, injection of thesense-strand control did too (9).This result was a puzzle until three years later. It was then that Fireand Mello first injected double-stranded RNA (dsRNA) — a mixtureof both sense and antisense strands — into C. elegans (Figure 2)(10). This injection resulted in much more efficient silencing thaninjection of either the sense or the antisense strands alone. This isprobably more info that you would like to look at, but I’ve insertedTable 1 from their paper, which shows of the effects that they sawfor different genes. In most cases, injection of sense or antisensehad little or no effect, but injection of dsRNA caused the samephenotype as mutants that had lost function in the gene beingtested.These investigators also showed that injection of dsRNA to aparticular gene reduced the levels of the endogenous transcripts.This is shown in Figure 3 for the mex-3 gene. These pictures are offour cell C. elegans embryos. A labeled antisense probe is used todetect the mex-3 transcript in these embryos. Panel (a) shows anembryo that wasn’t probed with antisense mex-3. Panel (b) showsan embryo probed with mex-3 antisense to detect mec-3 mRNA.The dark staining indicates that the cells are expressing lots of thetranscript. Panel (c) is an embryo that came from a mother that hadbeen injected with antisense mex-3. (Don’t get confused by the useof antisense used in the injection of the mother and the use of an5ʼ 3ʼ3ʼ 5ʼpar-1 geneAntisense par-1Injection producedmutant par-1 phenotypeSense par-1controlInjection producedmutant par-1 phenotypeFigure 2. The Guo & Kemphues experiment.antisense probe to detect the mRNA in the embryo.) You can seethat the level of mec-3 transcript is reduced compared with panel(b), but when dsRNA is injected into the mother her embyos (paneld) had no detectible mec-3 RNA when probed with antisense RNA.Thus, injection of dsRNA appeared to affect RNA stability.So how does injection of dsRNA lead to gene silencing? Manyresearch groups have diligently worked over the last few years toanswer this important question. A key finding by Baulcombe andHamilton provided the first clue. They identified RNAs of ~25nucleotides in plants undergoing cosuppression that were absent innon-silenced plants. These RNAs were complementary to both thesense and antisense strands of the gene being silenced (24).Further work in Drosophila — using embryo lysates and an in vitrosystem derived from S2 cells — shed more light on the subject (3,25, 26). In one notable series of experiments, Zamore andcolleagues found that dsRNA added to Drosophila embryo lysateswas processed to 21-23 nucleotide species. They also found
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