RNAi is a gene-silencing phenomenon triggered by double-stranded (ds) RNA and involves the generation of 21 to 26 nt RNA segments that guide mRNA destruction. In Caenorhabditis elegans, lin-4 and let-7 encode small temporal RNAs (stRNAs) of 22 nt that regulate stage-specific development. Here we show that inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-1), and two homologs of rde-1 (alg-1 and alg-2), cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further we show that dcr-1, alg-1, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7 stRNAs. Our findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation.

Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, we have selected C. elegans mutants resistant to dsRNA-mediated interference (RNAi). Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. We show that rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. We discuss implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing.

perhaps silencing tr&scriPtion at the &us. The alternative is that interference may prevent the processing or translation of the endogenous transcript. Several observations are most consistent with interference at a posttranscriptional step. First, only the sequences present in the mature transcript apDear to be effective at inducing interference. u e completion of the Caenorhabditis terference, two other remarkable features of Promoter and intron sequences appear to be elegans genome sequence represents a RNAi deserve comment. First is the obser-entirely ineffective (2). Second, some C. ele-T" major milestone in a journey initiated vation that the interfering activity can be guns genes exist in operons that are spliced by Sydney Brenner some 30 years ago. The transported across cell boundaries. Studies from a single transcript. If RNAi blocks goal then as now was to with dilute RNAs sug-transcription, then interfering with the 5' discover how genetic gest that the ideal tar-cistron would be expected to cause a polar information specifies get tissue for injec-effect that blocks the activity of all downthe development, anatotions is the intestine, stream cistrons. This does not appear to be my, and behavior of a even when the gene of the case. Several multicistronic genes have simple animal. Bringinterest is expressed in been analyzed (M), and in all cases, intering the full potential i f another tissue such as fering with the 5' cistrons can be accomthe genome sequence to bear on this goal will require facile new reverse genetic tools for converting sequence information into functional information. Here, we briefly describe progress toward understanding and using one such tool termed "RNA interference" or "RNAi." RNA interference was discovered by Guo and Kemphues (1) in the course of attempts to use antisense RNA to block gene expression in the maternal germ line. To their surprise, they found that both antisense and sense RNA preparations induced remarkably precise phenocopies of the targeted gene. Since then, both the efficacy and apparent lack of strand specificity associated with this interference process have been borne out in many subsequent studthe germ line or muscle (Fig. 1). Indeed, Lisa Timmons and Andrew Fire (4) have recently shown that feeding the worms Escherichia coli expressing the target gene dsRNA is sufficient to induce some interference. Thus, RNA uptake in the gut and distribution fiom the intestine to the somatic tissues and germ line can occur. Second, the RNAi effect is remarkably long lived. Potent plished without disturbing the expression of the downstream cistrons.Experiments with several maternal mRNAs suggest that RNAi does not destabilize or block the translation of the mature message. After RNAi injection into an adult hermaphrodite, we found that the first postinjection segment of the brood includes individuals that received both a functional interference is routinely observed not only maternal mRNA and the interfering RNA in the injected animal but also in all of the (5). In suc...

In Caenorhabditis elegans, the introduction of double-stranded RNA triggers sequence-specific genetic interference (RNAi) that is transmitted to offspring. The inheritance properties associated with this phenomenon were examined. Transmission of the interference effect occurred through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-1 and rde-4 were required for the formation of this interfering agent but were not needed for interference thereafter. In contrast, the rde-2 and mut-7 genes were required downstream for interference. These findings provide evidence for germ line transmission of an extragenic sequence-specific silencing factor and implicate rde-1 and rde-4 in the formation of the inherited agent.

The silencing of transgene expression at the level of transcription in the soma of Caenorhabditis elegans through an RNAi-dependent pathway has not been previously characterized. Most gene silencing due to RNAi in C. elegans occurs at the post-transcriptional level. We observed transcriptional silencing when worms containing the elt-2ϻgfp/LacZ transgene were fed RNA produced from the commonly used L4440 vector. The transgene and the vector share plasmid backbone sequences. This transgene silencing depends on multiple RNAi pathway genes, including dcr-1, rde-1, rde-4, and rrf-1. Unlike post-transcriptional gene silencing in worms, elt-2ϻgfp/LacZ silencing is dependent on the PAZ-PIWI protein Alg-1 and on the HP1 homolog Hpl-2. The latter is a chromatin silencing factor, and expression of the transgene is inhibited at the level of intron-containing precursor mRNA. This inhibition is accompanied by a decrease in the acetylation of histones associated with the transgene. This transcriptional silencing in the soma can be distinguished from transgene silencing in the germline by its inability to be transmitted across generations and its dependence on the rde-1 gene. We therefore define this type of silencing as RNAi-induced Transcriptional Gene Silencing (RNAi-TGS). Additional chromatin-modifying components affecting RNAi-TGS were identified in a candidate RNAi screen.[Keywords: RNAi; RNAi-TGS; RNAi-PTGS; dsRNA; siRNA; L4440] Supplemental material is available at http://www.genesdev.org.