Abstract
Dicer is a ribonuclease III-like enzyme playing a key role in the RNA silencing pathway. Genome sequencing projects have demonstrated that eukaryotic genomes vary in the numbers of Dicer-like (DCL) proteins from one (human) to four (Arabidopsis). Two DCL genes, MDL-1 and -2 (Magnaporthe Dicer-like-1 and -2) have been identified in the genome of the filamentous fungus Magnaporthe oryzae. Here we show that the knockout of MDL-2 drastically impaired gene silencing of enhanced green fluorescence protein by hairpin RNA and reduced related small interfering RNA (siRNA) accumulation to nondetectable levels. In contrast, mutating the other DCL, MDL-1, exhibited a gene silencing frequency similar to wild type and accumulated siRNA normally. The silencing-deficient phenotype and loss of siRNA accumulation in the mdl-2 mutant was restored by genetic complementation with the wild-type MDL-2 allele. These results indicate that only MDL-2 is responsible for siRNA production, and no functional redundancy exists between MDL-1 and MDL-2 in the RNA silencing pathway in M. oryzae. Our findings contrast with a recent report in the filamentous fungus Neurospora crassa, where two DCL proteins are redundantly involved in the RNA silencing pathway, but are similar to the results obtained in a more distantly related organism, Drosophila melanogaster, where an individual DCL protein has a distinct role in the siRNA/micro-RNA pathways.
Highlights
The ribonuclease III (RNase III)1 family, which processes double-stranded RNA precursors into mature RNAs, is one of the most interesting endonuclease families
Our findings contrast with a recent report in the filamentous fungus Neurospora crassa, where two DCL proteins are redundantly involved in the RNA silencing pathway, but are similar to the results obtained in a more distantly related organism, Drosophila melanogaster, where an individual DCL protein has a distinct role in the small interfering RNA (siRNA)/micro-RNA pathways
Do Multiple DCL Proteins Function Redundantly in the siRNA-mediated Gene Silencing Pathway Triggered by Hairpin RNA?—Small RNAs are regarded as sequence-specific guides in a variety of gene regulation processes including posttranscriptional mRNA degradation, translation inhibition, and modification of chromatin structure
Summary
The ribonuclease III (RNase III)1 family, which processes double-stranded RNA (dsRNA) precursors into mature RNAs, is one of the most interesting endonuclease families. Southern blots with EcoRI- and XbaI-digested genomic DNA and an EGFP probe indicated that the transcriptional units of both EGFP hairpin RNA and mRNA remained intact in the silenced transformants of mdl-1 and wild type.
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