RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21–25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. In this work, we analyzed cleavage of injected dsRNA to produce the initial siRNA population in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for organismal viability or fertility. Our results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger was comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduced the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs could circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed.

Posttranscriptional silencing of basic β-1,3-glucanase genes in the tobacco line T17 is manifested by reduced transcript levels of the gn1 transgene and homologous, endogenous basic β-1,3-glucanase genes. An RNA ligation-mediated rapid amplification of cDNA ends (RLM-RACE) technique was used to compare the 3′ termini of gn1 RNAs present in expressing (hemizygous and young homozygous) and silenced (mature homozygous) T17 plants. Full-length, polyadenylated gn1 transcripts primarily accumulated in expressing plants, whereas in silenced T17 plants, mainly 3′-truncated, nonpolyadenylated gn1 RNAs were detected. The relative abundance of these 3′-truncated gn1 RNA species gradually increased during the establishment of silencing in homozygous T17 plants. Similar 3′-truncated, nonpolyadenylated gn1 RNA products were observed in an independent case of β-1,3-glucanase posttranscriptional gene silencing. This suggests that these 3′-truncated gn1 RNAs are a general feature of tobacco plants showing posttranscriptional silencing of the gn1 transgene.