Molecular Cell, Vol. 6, 1077–1087, November, 2000, Copyright 2000 by Cell PressFunctional Anatomy of a dsRNA Trigger:Differential Requirement for the Two TriggerStrands in RNA Interference(PTGS) appear to be related or identical to dsRNA-asso-ciated RNA destabilization, although it is not clearwhether dsRNA is always the trigger (de Carvalho et al.,1992; Baulcombe, 1996; Vaucheret et al., 1998; Wasse-negger and Pellissier, 1998; Jorgensen et al., 1999). AsSusan Parrish,*†Jamie Fleenor,* SiQun Xu,*Craig Mello,‡and Andrew Fire*§* Carnegie Institution of WashingtonBaltimore, Maryland 21210†Biology Graduate ProgramJohns Hopkins University with the global (non-gene-specific) responses to dsRNAin mammalian cells, PTGS in plants has been identifiedBaltimore, Maryland 21218‡Program in Molecular Medicine as an antiviral mechanism (Baulcombe, 1999).The dsRNA-associated gene-specific responses ob-Department of Cell BiologyUniversity of Massachusetts Cancer Center served in plants and invertebrates are likely to involve(at some stage) the pairing of antisense RNA sequencesWorcester, Massachusetts 01605derived from the trigger with the endogenous senseRNA. (This feature is held in common by all modelsproposed to date for PTGS.) Antisense nucleic acidsSummaryhave long been known to be involved in specific cases ofphysiological regulation and to be applicable in certainIn RNA-mediated interference (RNAi), externally pro-vided mixtures of sense and antisense RNA trigger cases as tools for selective genetic disruption (Taka-yama and Inouye, 1990). The key (as yet unresolved)concerted degradation of homologous cellular RNAs.We show that RNAi requires duplex formation between questions in analysis of dsRNA-associated PTGS are(1) Why are both strands required in the trigger RNA?the two trigger strands, that the duplex must includea region of identity between trigger and target RNAs, and (2) How can dsRNA exert an effect at concentrationsthat are substantially lower than those of the endoge-and that duplexes as short as 26 bp can trigger RNAi.Consistent with in vitro observations, a fraction of in- nous target RNA? Several models have been proposedto explain the second observation, including the possi-put dsRNA is converted in vivo to short segments ofⵑ25 nt. Interference assays with modified dsRNAs in- bilities of multiround catalytic degradation of targetRNAs using a denatured region of the double-strandeddicate precise chemical requirements for both basesand backbone of the RNA trigger. Strikingly, certain trigger RNA (Montgomery et al., 1998) or production ofnumerous short antisense RNA copies of the incomingmodifications are well tolerated on the sense, but notthe antisense, strand, indicating that the two trigger trigger RNA (Wassenegger and Pellissier, 1998).Genetic screens for components necessary for PTGS/strands have distinct roles in the interference process.RNAi have been one approach toward identifying molec-ular components of the mechanism (e.g., Cogoni andIntroductionMacino, 1999; Tabara et al., 1999; Dalmay et al., 2000;Mourrain et al., 2000). While the cloned genes and mu-Double-stranded RNA (dsRNA) induces potent cellularresponses in diverse biological systems (Fire, 1999; tant strains provide hints and useful tools for futurestudies, direct biochemical roles for the genetically iden-Sharp, 1999; Williams, 1999). Models to explain dsRNAresponses have centered on possible defense against tified PTGS factors have yet to be assigned.A complementary approach toward understandingdeleterious RNAs, including viral transcripts and replica-tion intermediates (Ratcliff et al., 1997; Williams, 1999) PTGS comes from attempts to understand the chemicalcharacter of the trigger RNA that is critical for inducingand transposons (Ketting et al., 1999; Tabara et al.,1999). In mammalian cells, dsRNA is associated with a interference. The nematode system offers a several ad-vantages for this analysis, since small quantities of syn-sequence-nonspecific response that includes inductionof interferon, phosphorylation of translation initiation thetic trigger RNAs can be assayed for interference ac-tivity in vivo by a straightforward injection assay. Thisfactor eIF2 (which leads to a general block in translation),and induction of a 2⬘-5⬘ oligoadenylate synthetase provides a sensitive means to test model substrates andto analyze the effects of various perturbations to the(which can stimulate the RNA degrading enzyme RNaseL) (Williams, 1999). RNA trigger. In this paper, we investigate the chemicaland sequence requirements for RNA-triggered gene si-A distinct type of response (termed RNAi) has beenassociated with dsRNA in numerous species of inverte- lencing in C. elegans.brates, plants, and protozoa (for reviews, Fire, 1999;Sharp, 1999). In these cases, the response to dsRNAResultsincludes a dramatic and sequence-specific destabiliza-tion of cellular RNA transcripts that correspond to theRequirements for Length and SequenceRNA trigger.Composition of the RNAi TriggerA subset of the transgene-triggered processes re-From first principles, it was conceivable that RNA-medi-ferred to in plants as posttranscriptional gene silencingated interference required a specific sequence in theinterfering RNA or target RNA. The ability to target largenumbers of different genes in C. elegans for interference§To whom correspondence should be addressed (e-mail: [email protected]).suggests that any such sequence must be common inMolecular Cell1078Figure 1. Sequence and Size Requirementsfor the RNA Trigger(A) A series of nonoverlapping fragments ofthe gfp coding region. dsRNAs correspond-ing to each fragment were prepared enzymat-ically and injected at concentrations in therange of 20–50 ␮g/ml into transgenic animalscarrying a myo-3::gfp transgene (PD4251, seeExperimental Procedures). Interference lev-els (loss of GFP) were quantitated as de-scribed in Experimental Procedures. Slightdifferences in potency between the shortRNAs could reflect some variation in concen-tration or purity between RNA preparations.(B) Five short dsRNA sequences from theunc-22 coding region that were prepared syn-thetically. Fractions shown are numbers of F1progeny following injection that twitched in0.3 mM levamisole. Synthetic sense and anti-sense strands were annealed and injectedinto wild-type C. elegans (concentrations 9.5mg/ml [zr7, zr9, zr11]; 4.8 mg/ml [zr1]; 2.3 mg/ml [zr3]. Additional injections of zr11 at 19mg/ml showed no