transgene-induced silencing have been demonstrated to act post-transcriptionally (9). In addition to the four mRNAs, we also tested the accumulation of a potential RNA transcript corresponding to an intergenic region, which produced less esRNA in dcl strains than in wild type. A transcript was detected from this intergenic locus and it also showed a higher accumulation in the dcl2 single and dcl1 /dcl2 double mutants relative to the wild type (Figure 2, locus 3), supporting that the diﬀerent esRNAs of Mucor are functional. Biogenesis of exonic endogenous siRNAs Next, we wanted to understand the mechanism that produced ex-siRNAs. Since most exons, which generated less ex-siRNAs in dcl2 than in wild type, produced some antisense ex-siRNAs we hypothesised that an RdRP is involved in ex-siRNAs biogenesis. Two diﬀerent RdRP proteins have been implicated in M. circinelloides transgene-induced gene silencing (S.C., S.T-M., R.M.R-V, our unpublished results), and we tested the in- volvement of these proteins, RdRP1 and RdRP2, in the biogenesis of ex-siRNAs by deep sequencing of esRNAs from rdrp1 and rdrp2 strains. The normalized reads of ex-siRNAs from the 324 exons that accumulated less ex-siRNA in, at least, one dcl mutant compared to the wild-type strain are shown in Supplementary Table S2, which also shows the strand bias (sense/antisense) of the ex-siRNAs produced by the wild-type and all mutant strains. The accumulation of ex-siRNAs from each exon in the diﬀerent mutant strains was compared to the wild type and the fold diﬀerence is shown in Supplementary Table S3, in which exons are ordered according to the log2 fold change of ex-siRNAs in the dcl2 strain Table 3. Characteristics of the four classes of ex-siRNA Nucleic Acids Research, 2010, Vol. 38, No. 16 5539 compared to wild type. Four diﬀerent groups can be easily identiﬁed and we call these almost perfectly separated groups class 1–4. Table 3 shows a summary of the properties of the diﬀerent ex-siRNA classes. Classes 1 and 2 include all ex-siRNAs that are dcl2-de- pendent. The ﬁrst nucleotide of these ex-siRNAs exhibits a strong preference for uracil (92%; Table 3; Supplementary Figure S3). This preference was also shown for Argonaute-bound guide RNAs of animal, plants and other fungi (16). The majority of the DCL2-dependent ex-siRNAs also showed reduced levels in the rdrp1 mutant but not in the rdrp2 strain and deﬁne class 2. This is the largest group of ex-siRNAs, with 222 exons that represent 68.5% of the 324 ex-siRNA producing exons. Most likely, these ex-siRNAs act in cis, since mRNAs transcribed from these exons are up-regulated in the absence of the ex-siRNAs (Figure 2). There is not a strong strand bias among these ex-siRNAs, most exons producing a mixed sense and antisense ex-siRNAs, which is expected considering that a large number of ex-siRNAs are produced from each exon (Supplementary Figure S1). The requirement for RdRP1 and DCL2 for the biogenesis of this ex-siRNA class suggests that mRNAs from those loci are turned into dsRNA by RdRP1 and then processed by DCL2. The involvement of these two proteins in the biogenesis of the majority of ex-siRNAs can be extended to esRNAs derived from transposons and intergenic regions because most of the dcl2-dependent esRNAs generated from those loci also require rdrp1 (Figure 3). A very small group of the dcl2-dependent ex-siRNAs, made up by ex-siRNAs from only nine exons belonging to ﬁve genes, does not require RdRP1 but most of them depend on RdRP2. These ex-siRNAs (class 1) are the sRNA class Strand bias Average log2 fold change from WT No. of exons ex-siRNA (%) 50 Ua (%) 30 penult. Ub (%) Class 1 Class 2 Class 3 Class 4 0.78 0.34 0.90 0.83 dcl1 dcl2 Dcl1 /2 rdrp1 rdrp2 0.34 0.55 0.47 2.45 12.84 3.79 0.12 0.26 8.86 8.72 3.21 1.02 3.74 5.02 4.52 2.61 1.33 1.36 3.50 2.42 9 222 88 5 13.42 58.74 27.36 0.48 92.18 92.12 8.39 28.28 6.11 16.63 49.57 66.61 Diﬀerent characteristics of ex-siRNAs from each classes are shown in the table. Strand bias indicates orientation to mRNAs, where 1 corresponds to all sRNAs in the same orientation as the mRNA, 0 to equal mixture of sRNAs on both strands and 1 to all sRNAs antisense to mRNAs. Numbers in bold indicate a higher than 4-fold down-regulation in the corresponding mutants relative to wild type. aThe percentage of redundant reads that contain a uracil in the 50 most position. bThe last column shows the percentage of redundant reads that contain a uracil in the 30 penultimate position. Figure 3. rdrp1 and rdrp2 dependence of DCL2 generated esRNAs. The pie charts show the percentage of DCL2-dependent loci that also show reduced level of esRNAs in rdrp1 and rdrp2 strains.