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Abstract
RNA interference (RNAi) in animals, cosuppression in plants, and quelling in fungi are homology-dependent gene silencing mechanisms in which the introduction of either double-stranded RNA (dsRNA) or transgenes induces sequence-specific mRNA degradation. These phenomena share a common genetic and mechanistic basis. The accumulation of short interfering RNA (siRNA) molecules that guide sequence-specific mRNA degradation is a common feature in both silencing mechanisms, as is the component of the RNase complex involved in mRNA cleavage. During RNAi in animal cells, dsRNA is processed into siRNA by an RNase III enzyme called Dicer. Here we show that elimination of the activity of two Dicer-like genes by mutation in the fungus Neurospora crassa eliminates transgene-induced gene silencing (quelling) and the processing of dsRNA to an siRNA form. The two Dicer-like genes appear redundant because single mutants are quelling proficient. This first demonstration of the involvement of Dicer in gene silencing induced by transgenes supports a model by which a dsRNA produced by the activity of cellular RNA-dependent RNA polymerases on transgenic transcripts is an essential intermediate of silencing.
We thank Tony Nolan for critical reading of the manuscript; Robert Metzenberg and Patrick Shiu for help in crossing the mutants; Marina Goldoni, Laura Braccini, and Gianluca Azzalin for help with some experiments; Peng Fang and Cheng Wu for help with extract preparation; and the Whitehead Institute for access to the Neurospora genome database.
Caterina Catalanotto is a recipient of a CIB (Consorzio Interuniversitario per le Biotecnologie) fellowship. This work was supported by CNR MIUR Progetto Strategico (grant 02.00644.ST97), by FIRB (grant RBNE015MPB_001/RBNE01KXC9_006) and by the NIH (grant GM47498).