5538 Nucleic Acids Research, 2010, Vol. 38, No. 16 Figure 1. Accumulation of esRNAs in wild type and dcl mutant Mucor strains. Northern blots of esRNAs from intergenic regions (A) and exons of protein coding genes (B). Low-molecular weight RNA was extracted from wild-type, dcl1 , dcl2 and dcl1 /dcl2 double mutant strains, separated on 15% denaturing polyacrylamide gels, transferred to membranes and probed with riboprobes or end-labelled DNA oligo- nucleotides speciﬁc to each locus. For exact probe sequences, see Supplementary Table S1. Ethidium bromide stained images of gels below the radiograms show equal loading of lanes. (B) The accumula- tion of antisense and sense esRNAs separately. The exon loci corres- pond to the following proteins: locus 4: ID 80452, serine/threonine kinase; locus 5: ID 82197, no domains found; locus 6: ID 77050, no domains found; locus 7: ID 78553, low similarity to transposase 21 protein; locus 8: ID 85423, Zn-ﬁnger CCHC containing protein; locus 9: ID 95230, no domains found. Ten picomoles per lane of 23-mer to 27-mer DNA oligonucleotides in antisense and sense orientation were used as size markers and to control the hybridization speciﬁcity. In all cases, the RNA probes hybridized to these controls. Sometimes diﬀerent sized ex-siRNAs were detected from the same locus, which seems to be a characteristic of the M. circinelloides DCL enzymes. In fact, transgene-induced gene silencing is also associated with two diﬀerent size Figure 2. Accumulation of mRNAs in wild type and dcl mutant Mucor strains. Northern blots of high molecular weight RNAs corresponding to an intergenic region (locus 3) or protein coding exons (loci 4, 5, 8 and 9). Total RNA (50 mg) extracted from wild-type and mutant strains were separated in 1.2% denaturing agarose gel, transferred to mem- branes and hybridized with gene speciﬁc or rRNA probes (Supplementary Table S1). The locus numbers correspond to the loci on Figure 1. classes of siRNAs, 21 and 25 nt long, both in the wild-type and the dcl1 mutant (9,10). Accumulation of ex-siRNAs does not necessarily mean that they are functional, as for example some non-conserved plant miRNAs do not have target mRNAs (17). To test whether the exon mapping Mucor ex-siRNAs aﬀect gene expression, accumulation of the mRNAs was analysed by northern blot analysis. All the four tested mRNAs that showed reduced ex-siRNA ex- pression in dcl2 accumulated at an increased level in the dcl mutant strains compared to the wild type (Figure 2, loci 4–9) conﬁrming that the Mucor ex-siRNAs are functional. The eﬀect of ex-siRNAs on mRNA accumulation is most likely post-transcriptional, since no signiﬁcant methylation is associated with gene silencing in Mucor, and siRNAs generated in