Table 2. Number of loci down-regulated at sRNA level in dcl1 and dcl2 mutants Type of loci Transposons Intergenic regions Exons Total Down-regulated in dcl1 0 (0) 7 (2) 5 (5) 12 (7) dcl2 207 (207) 401 (396) 225 (225) 833 (828) dcl1 /dcl2 205 (2) 441 (44) 319 (94) 965 (140) All dcl 209 447 324 980 Number of loci (with normalized abundance of more than 50 reads per million) showing a fourfold or higher reduction in the diﬀerent mutant strains compared to the wild type. Numbers in parentheses in the dcl1 and dcl2 columns show the number of loci that require only that Dicer. The numbers in parentheses for double mutant shows the number of loci that are down-regulated only in the double mutant (the number of loci that can be processed by either Dicer). Please note that the four transposon loci that are not down-regulated in dcl1 /dcl2 but reduced in dcl2 , are just below the 4-fold threshold in the double mutant. Reads that mapped to the genome in close proximity ( 200 bp) to each other were grouped together into loci (as previously described in ref. 13). A total of 24 111 loci were identiﬁed all over the Mucor genome by this criter- ion. All esRNA producing loci were analysed by comparing accumulation of esRNAs in the wild-type and dcl mutants. Many loci produced esRNAs only in sense orientation at a similar rate in wild-type and all mutant strains, and accumulation of esRNAs from some of these loci was analysed by northern blot. These esRNAs were either not detectable or the probes detected a smear between 15 and 2000 nt but not bands with distinct sizes between 20 and 25 nt (data not shown). Therefore, we concluded that reads from these loci are most likely to be degradation products of ribosomal, transfer and mes- senger RNAs and were not further analysed. To identify DCL-dependent esRNA loci, we selected those that showed at least a 4-fold decrease in normalized esRNA reads in dcl1 or dcl2 mutants compared to wild type. Eight hundred and forty diﬀerentially expressed loci were identiﬁed, which were grouped based on the annota- tion of the locus: intergenic, transposon or exon (Table 2). Seven diﬀerentially expressed loci showed a decrease only in dcl1 , 828 were down-regulated in only dcl2 and ﬁve were suppressed in both. Next, we compared esRNA ac- cumulation in wild-type and dcl1 /dcl2 double mutant. Most of the 840 loci producing fewer esRNAs in either dcl1 or dcl2 or in both also produced fewer esRNAs in the double mutant. In fact, the read numbers were usually lower in the double mutant than in any of the single mutants, demonstrating some redundancy for the two dcl genes (Supplementary Figure S1). In addition a further 140 loci showed diﬀerential expression only in the double mutant making the total number of dcl (dcl1 and/or dcl2) dependent esRNA producing loci 980. Despite the redundancy between dcl1 and dcl2, the sequencing data suggested that DCL2 is the primary protein that generates the majority of esRNAs in Mucor, although from a very small number of loci mainly DCL1 produces esRNAs and there are loci from which both Nucleic Acids Research, 2010, Vol. 38, No. 16 5537 Dicers can make esRNAs. The DCL proteins have similar hierarchy in transgene-induced silencing, where DCL2 also plays a more prominent role in the generation of siRNAs than DCL1 (8). Short RNAs are produced from transposons but there is no evidence for miRNA genes In order to investigate the distribution of DCL-dependent esRNAs across the diﬀerent types of loci, we calculated the normalized abundance of esRNAs per 1 kb of exonic, intergenic and transposon regions in the wild-type and dcl mutant strains. Supplementary Figure S2 shows that esRNAs are not formed at random across the genome but they are enriched in exonic regions compared with intergenic regions and transposons. In fact, only 209 out of the 980 esRNA producing loci, down-regulated in at least one of the dcl strains, were annotated transposons or repeats (Table 2). This is in contrast with other organ- isms, such as Schizosaccharomyces pombe (15), Saccharomyces castellii and Candida albicans (16), where most esRNAs are produced from repeats and transposons. Intergenic and intronic loci are the prime candidates for producing miRNAs. Therefore, we asked whether any of these loci can be folded into a stem–loop structure char- acteristic of miRNA loci. None of the 447 dicer-dependent intergenic and intronic loci fulﬁlled the criteria of miRNA loci, although several esRNAs from intergenic regions were conﬁrmed by northern blot (Figure 1A). We also tested all 24 111 esRNA producing loci identiﬁed in the initial analysis (including those that are not down-regulated in dcl mutants) but none of them had the features of miRNA genes. It is likely that miRNAs are not present in Mucor, although we cannot exclude the possibility that they are expressed in diﬀerent growth condition or developmental stages. The apparent lack of miRNAs in fungi and the presence of transposon derived esRNAs in basal fungi, plants and animals suggest that life evolved esRNAs to silence transposons and than each branch of life has utilised this machinery to silence other targets. Short RNAs are generated from exons and regulate mRNA accumulation Next, we focussed on the main class of esRNAs that mapped to exons (Table 2 and Supplementary Figure S2) and we call these exonic-siRNAs (ex-siRNAs) based on their unusual location. In total, 324 exonic loci were identiﬁed, which correspond to 276 genes, since some genes contain more than one exon. To validate the sequencing data, accumulation of selected ex-siRNAs was analysed by northern blot (Figure 1B), and in all cases the presence of distinct bands around 20–25 nt was conﬁrmed. Some loci produced almost exclusively anti- sense ex-siRNAs, whereas others produced ex-siRNAs in both orientations. Results also conﬁrmed the reduced ex- pression of ex-siRNAs in mutant strains compared to wild type, as well as that DCL2 is the primary dicer in Mucor. The northern blots corroborated the redundancy between the two dcl genes because the ex-siRNA signals were often weaker in the double mutant than in the single mutants.