Redundancy of the two dicer genes in transgene-induced posttranscriptional gene silencing in Neurospora crassa

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.

FIG.1.

dcl-1 and dcl-2 knockouts. (A) Schematic depiction of dcl-1 and dcl-2 loci before and after homologous recombination with a linear DNA molecule. White box, genomic dcl-1 and dcl-2 loci; black box, hygromycin B resistance cassette; grey box, region of homology between dcl gene flanking sequences and the branches of the linear DNA; up1 and up2, sequences upstream of dcl-1 and dcl-2 loci, respectively; down1 and down2, sequences downstream of dcl-1 and dcl-2 loci, respectively; arrows, restriction sites for XhoI and BamHI; solid lines, predicted bands; dashed lines, ³²P-labeled probes. (B) Southern blot analysis of WT and four independent dcl-1 transformants. Genomic DNA was digested with XhoI and hybridized with a DNA probe complementary to the upstream region of the dcl-1 gene. Transformant in lane 12 shows a band of ∼2.4 kb predicted for the dcl-1 knockout. (C) Southern blot analysis of WT and eight independent dcl-2 transformants. Genomic DNA was digested with BamHI and hybridized with DNA complementary to the upstream region of the dcl-2 gene. Transformants in lanes 6 and 19 show a band of ∼2.0 kb predicted for the dcl-2 knockout. (D) Identification of the dcl-1/dcl-2 double mutant. The contemporaneous presence of dcl-1 and dcl-2 mutations was monitored by a double Southern analysis of genomic DNAs from the WT strain and five ascospores originating from a cross between dcl-1⁻/helper and dcl-2⁻ strains. The Southern blotting conditions used to identify both the dcl-1⁻ locus and dcl-2⁻ locus are as reported in Materials and Methods. The dashed arrows point to the predicted sizes of the dcl-1 and dcl-2 knockouts. The stars indicate the double mutant strain.

FIG. 2.

Dicer activity in N. crassa extracts. Denaturing acrylamide gel analysis of the dsRNA cleavage assay of different N. crassa strain lysates incubated with ³²P-radiolabeled double-stranded molecules for the times indicated. (A) The WT lysate was incubated in the presence (+) or absence (−) of an energy regeneration system. (B) ³²P-radiolabeled dsRNA was incubated with protein extracts from the WT, a quelled strain, and the qde-1⁻, qde-2⁻, and qde-3⁻ mutant strains, and the progression of the reactions was monitored at different times. The size of the RNA was estimated by using 25-nt DNA oligonucleotides as size markers.

FIG. 3.

Dicer domains in N. crassa Dicer proteins. Black bars correspond to the full protein sequence. The boxes correspond to the identified domains, each with its starting and stopping amino acid. Both DCL1 and DCL2 of Neurospora contain a DEAD box, a helicase C domain (hel C), a duf283 domain with an unknown function, and two RNase III domains (RNase IIIa and RNase IIIb). DCL2 contains also a dsRNA binding domain. aa, amino acids.

FIG. 4.

dsRNA and siRNAs present in a double Dicer mutant strain. (A) Northern blot of total RNAs extracted from the DT1 transformant, WT, and a heterokaryotic strain (DT1 plus WT) made by using a sense riboprobe corresponding to the al-1 sequence. (B) Northern blot of low-molecular-weight enriched RNA preparations from strains as indicated in panel A made by using a hydrolyzed riboprobe corresponding to the al-1 gene.

FIG. 5.

Dicer activity in N. crassa with analysis of WT, dcl-1, dcl-2, and dcl-1/dcl-2 strains. Cell extracts were incubated with radiolabeled dsRNA for 0, 30, or 90 min (T₀, T₃₀, and T₉₀, respectively) in the presence (+) or absence (−) of an energy regenerating system as indicated, and the RNA was examined by denaturing gel electrophoresis as described in Materials and Methods. Decade RNA markers (Ambion) labeled with ³²P were used as size standards (M).