Abstract
RNA interference (RNAi) is induced by the direct transfer of double-stranded RNAs (dsRNAs) into protoplasts prepared from Arabidopsis thaliana seedlings. In this protoplast RNAi system, we compared the efficacies of various-sized dsRNAs (between 21 and 139 nucleotides [nt]) for inducing RNAi and assessed the dsRNA-cleaving activities of Dicer-like 3 (DCL3) and 4 (DCL4). After the direct transfer of dsRNAs into protoplasts, cleaved RNA products of 21 nt were detected from long 130- or 500-nt dsRNAs by DCL4 but not from 37-nt dsRNAs. These results indicate that DCL4 preferentially cleaves long dsRNAs in protoplasts, consistent with our previous biochemical data regarding the substrate specificity of DCL4. Direct transfer of long dsRNAs of approximately 130 nt into protoplasts induces RNAi much more effectively (by approximately 60- to 400-fold) than direct transfer of short 37-nt dsRNAs. Although transfer of 21-nt dsRNAs into protoplasts induced RNAi without DCL4 activity, the induction of RNAi was less effective (by approximately 0.01-fold) compared with long dsRNAs. These results indicate that cleavage of long dsRNAs exceeding 100 nt by DCL4 into 21-nt dsRNAs is essential for efficient induction of RNAi in plant cells.
Highlights
RNA interference (RNAi) is the process of sequence-specific post-transcriptional gene silencing (PTGS) triggered by double-stranded RNAs[1,2]
These results indicate that DCL4 preferentially cleaves long double-stranded RNAs (dsRNAs) (>100 nt) into 21-nt RNAs in protoplasts, confirming our previous finding demonstrating that DCL4 preferentially cleaves long dsRNAs into 21-nt dsRNAs in vitro[21]
We demonstrated for the first time that long DCL4-substrate dsRNAs >130 nt induce RNAi up to 400-fold more potently than short dsRNAs of 21 or 37 nt and that the cleavage of long dsRNAs by DCL4 is essential for efficient induction of RNAi in plant cells (Supplementary Fig. S1)
Summary
RNA interference (RNAi) is the process of sequence-specific post-transcriptional gene silencing (PTGS) triggered by double-stranded RNAs (dsRNAs)[1,2]. Most eukaryotic organisms except for mammals, including fungi, insects, and plants, which have no antibody-mediated immune system, sense long exogenous dsRNAs as viruses and activate the RNAi (PTGS) pathway for defence against virus infection[6,7,8]. Kim et al demonstrated that dsRNAs of 25 to 30 nt in length induce RNAi as much as 100-fold more potently than 21-nt dsRNAs (siRNA duplexes), and this greater potency depends on processing of the 25- to 30-nt dsRNAs by Dicer[13]. They termed these dsRNAs “Dicer-substrate” siRNAs (dsiRNAs). Even though some important genes encoding RNAi machinery components were first discovered in the model plant A. thaliana via genetic studies[15,16], findings from biochemical studies related to the molecular mechanism of plant RNAi are limited
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