RNA interference So that I don t get myself into any trouble most of the following text is verbatim from the Ambion website which nicely summarizes a lot of what I will talk about in class I ve edited it a bit and pasted in some figures to help but I m not keeping tract of what the website had and what I ve inserted If you want to see the original Ambion text go to http www ambion com techlib hottopics rnai The first experiments Post transcriptional gene silencing PTGS which was initially considered a bizarre phenomenon limited to petunias and a few other plant species is now one of the hottest topics in molecular biology 1 In the last few years it has become clear that PTGS occurs in both plants and animals and has roles in viral defense and transposon 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 a variety of organisms reviewed in 1 3 How was RNAi discovered How does it work Perhaps more importantly how can it be harnessed for functional genomics experiments This article will briefly answer these questions and provide you with resources to find in depth information on PTGS and RNAi research Figure 1 The attempt to overexpress chalone synthetase an enzyme that produces anthocyanin pigment resulted not in a darker petunia flower but instead a loss of flower pigment More than a decade ago a surprising observation was made in petunias While trying to deepen the purple color of these flowers Rich Jorgensen and colleagues introduced a pigment producing gene under the control of a powerful promoter Instead of the expected deep purple color many of the flowers appeared variegated or even white Jorgensen named the observed phenomenon cosuppression since the expression of both the introduced gene and the homologous endogenous gene was suppressed 1 5 First thought to be a quirk of petunias cosuppression has since been found to occur in many species of plants It has also been observed in fungi and has been particularly well characterized in Neurospora crassa where it is known as quelling 1 3 But what causes this gene silencing effect Although transgeneinduced silencing in some plants appears to involve gene specific methylation transcriptional gene silencing or TGS in others silencing occurs at the post transcriptional level posttranscriptional gene silencing or PTGS Nuclear run on experiments in the latter case show that the homologous transcript is made but that it is rapidly degraded in the cytoplasm and does not accumulate 1 3 6 Introduction of transgenes can trigger PTGS however silencing can also be induced by the introduction of certain viruses 2 3 Once triggered PTGS is mediated by a diffusible trans acting molecule This was first demonstrated in Neurospora when Cogoni and colleagues showed that gene silencing could be transferred between nuclei in heterokaryotic strains 1 7 It was later confirmed in plants when Palauqui and colleagues induced PTGS in a host plant by grafting a silenced transgene containing source plant to an unsilenced host 8 From work done in nematodes and flies we now know that the trans acting factor responsible for PTGS in plants is dsRNA 1 3 RNAi Is Discovered in Nematodes The first evidence that dsRNA could lead to gene silencing came from work in the nematode Caenorhabditis elegans Seven years ago researchers Guo and Kemphues were attempting to use antisense RNA to shut down expression of the par 1 gene in order to assess its function As expected injection of the antisense RNA disrupted expression of par 1 but quizzically injection of the sense strand control did too 9 par 1 gene Antisense par 1 3 Sense par 1control 5 Injection produced mutant par 1 phenotype 5 3 Injection produced mutant par 1 phenotype Figure 2 The Guo Kemphues experiment This result was a puzzle until three years later It was then that Fire and Mello first injected double stranded RNA dsRNA a mixture of both sense and antisense strands into C elegans Figure 2 10 This injection resulted in much more efficient silencing than injection of either the sense or the antisense strands alone This is probably more info that you would like to look at but I ve inserted Table 1 from their paper which shows of the effects that they saw for different genes In most cases injection of sense or antisense had little or no effect but injection of dsRNA caused the same phenotype as mutants that had lost function in the gene being tested These investigators also showed that injection of dsRNA to a particular gene reduced the levels of the endogenous transcripts This is shown in Figure 3 for the mex 3 gene These pictures are of four cell C elegans embryos A labeled antisense probe is used to detect the mex 3 transcript in these embryos Panel a shows an embryo that wasn t probed with antisense mex 3 Panel b shows an embryo probed with mex 3 antisense to detect mec 3 mRNA The dark staining indicates that the cells are expressing lots of the transcript Panel c is an embryo that came from a mother that had been injected with antisense mex 3 Don t get confused by the use of antisense used in the injection of the mother and the use of an antisense probe to detect the mRNA in the embryo You can see that the level of mec 3 transcript is reduced compared with panel b but when dsRNA is injected into the mother her embyos panel d 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 Many research groups have diligently worked over the last few years to answer this important question A key finding by Baulcombe and Hamilton provided the first clue They identified RNAs of 25 nucleotides in plants undergoing cosuppression that were absent in non silenced plants These RNAs were complementary to both the sense and antisense strands of the gene being silenced 24 Further work in Drosophila using embryo lysates and an in vitro system derived from S2 cells shed more light on the subject 3 25 26 In one notable series of experiments Zamore and colleagues found that dsRNA added to Drosophila embryo lysates was processed to 21 23 nucleotide species They also found that the homologous endogenous mRNA was cleaved only in the region corresponding to the introduced dsRNA and that cleavage occurred at 21 23 nucleotide intervals 26
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