In numerous organisms the introduction dsRNA can induce sequence specific post-transcriptional silencing (PTGS) of a corresponding gene. The experimental application of dsRNA to induce gene silencing has been termed RNA interference or RNAi. Although RNAi has gained wide acceptance in C. elegans , Drosophila and several other organisms as a tool for silencing genes, there is, as yet, relatively little known about the mechanism and the underlying physiological role of RNAi in the organism. Importantly, it is not yet known if mechanisms related to RNAi have a role in the natural developmental regulation of gene expression. One of the genes necessary for RNAi in C.elegans,
rde-1 , belongs to an ancient gene family with relatives in numerous eukaryotic organisms including plants, Neurospora, Drosophila and mammals. Although
rde-1 mutants do not have obvious developmental defects, its relative,
ago1 , which is involved in PTGS in Arabidopsis was initially found as a gene important for growth and proper morphogenesis of the plant . The
rde-1 family in C.elegans contains more than 20 members with two predicted genes that are very similar to the plant gene
ago1 . Members of a second gene family including the plant gene carpel factory (
caf1 ) and the fly gene dicer encode multifunctional proteins that share several domains including a motif found in RDE-1 as well as RNase III, RNA helicase and dsRNA binding domains. Mutations in
caf1 cause developmental abnormalities not unlike those seen in
ago1 mutants. Drosophila dicer , has recently been shown to play role in processing a trigger dsRNA into small 22 nucleotide (nt) interfering RNAs important for RNAi. We used RNAi to target the
ago1 related genes which we call
alg-1 and
alg-2 ( a rgonaute l ike g enes) as well as the C. elegans homolog of
caf1/dicer which we call
dcr-1 . Our analysis revealed developmental phenotypes reminiscent of the heterochronic mutant phenotypes displayed by
lin-4 and
let-7 loss of function mutants.
lin-4 and
let-7 encode small ~22 nt RNAs that regulate stage specific gene expression by pairing with complementary sequences in the 3' UTRs of their target genes
lin-14 and
lin-41 . Genetic analysis suggests that defects associated with
alg-1 and
dcr-1(RNAi) are caused at least in part by misregulation of
lin-14 and
lin-41 . The RNA products of
lin-4 and
let-7 are predicted to be transcribed as ~70 nt precursor RNAs that fold into comparable stem-loop structures. Such RNA precursors are detected by Northern analyses of C. elegans RNA. Additionally,
let-7 homologues exist in numerous metazoan species and
let-7 precursor RNAs are observed in Drosophila and humans, supporting the conservation of a common secondary structure as well as the major ~22 nt
let-7 RNA sequence. In order to examine
lin-4 and
let-7 RNA expression we prepared RNA from L3/L4 staged progeny of
alg-1 and
dcr-1(RNAi) animals. In
dcr-1(RNAi) animals we observed a significant accumulation of the 70 nucleotide forms of
lin-4 and
let-7 transcripts. A similar accumulation of the large form of
lin-4 but not
let-7 was observed in
alg-1(RNAi). These findings would be consistent with a model in which the longer forms of
lin-4 and
let-7 are processed by DCR-1 into ~22 nt products. Interestingly, we find that
dcr-1(RNAi) also appears to cause a significant reduction in RNAi itself. Thus, DCR-1 may have similar roles in processing dsRNA into small RNAs that mediate both developmental regulation and RNA interference. In contrast, RDE-1 and its homologues ALG-1 and ALG-2 appear to be dedicated to distinct functions. In the future it will be fascinating to learn more about the shared features of these mechanisms as well as how RDE-1 homologues and other factors specify apparent differences in the outcomes of these pathways: RNA destruction versus translation inhibition.