5540 Nucleic Acids Research, 2010, Vol. 38, No. 16 most down-regulated in the dcl2 strain (Supplementary Table S3). Accumulation of these ex-siRNAs is strongly up-regulated in the rdrp1 mutant, which may suggest that both RdRPs can compete for binding the mRNA tem- plates but only RdRP2 can turn them into dsRNA. Consequently, lack of RdRP1 may allow RdRP2 to make more dsRNA that would result in a higher ex-siRNA level. We have investigated the biological functions of the genes that produce less ex-siRNAs in the dcl2 mutant (classes 1 and 2), trying to link those functions with the phenotype of the dcl2 mutant strain (8). However, the high number of genes aﬀected in the dcl2 mutant makes it dif- ﬁcult to understand their roles and to reveal their biologic- al signiﬁcance. Nonetheless, it can be emphasized that many of those genes are annotated as encoding for proteins involved in signal transduction and information storage and processing (Supplementary Table S3), which may indicate the involvement of these ex-siRNAs in the regulation of diﬀerent cellular processes. A signiﬁcant group of ex-siRNAs producing exons (88 out of 324) are in class 3 and correspond to ex-siRNAs that are down-regulated only in the double dicer mutant but not in dcl1 or dcl2 single mutants (Supplementary Table S3). This indicates that dsRNA produced from these exons can be processed by either DCL1 or DCL2. However, both RdRP enzymes are involved in the biogenesis of these ex-siRNAs, because they are down-regulated in the absence of either of the two rdrp genes. A peculiar feature of the class 3 ex-siRNAs is that they show a very strong strand bias, almost all of them are exclusively sense to the mRNAs. Besides that, the class 3 ex-siRNA molecules show a random spread of size distribution that is not observed in the dcl2 dependent classes 1 and 2, which produce predominantly 23–24 nt sRNAs (Supplementary Figure S4). This suggests that class 3 ex-siRNAs are not produced by a canonical RNA silencing mechanism. Instead, we can speculate that sequential or combined activity of RdRP1 and RdRP2 generates dsRNA stretches but not dsRNA through the entire length of the mRNA. These discrete dsRNA regions are processed by either DCL1 or DCL2 and after the initial cleavage, the single stranded portions of mRNAs are degraded by non-speciﬁc RNases, probably because they lose their cap and/or polyA tail. Alternatively, DCL1 or DCL2 may process these mRNAs beyond the discrete dsRNA regions, since the ability of human Dicer to cleave ssRNA in a partial dsRNA molecule has been described recently (18). We also analysed the nucleotide distribution in each position of class 3 ex-siRNAs and found a very strong bias for uracil in the penultimate position for almost all sizes of short RNAs (20–25 nt; Supplementary Figure S5). This is very surprising considering that uracil is strongly under-represented in all esRNAs (including class 3 ex-siRNAs; Supplementary Table S4). The strong bias for uracil in the penultimate position suggests that the generation of class 3 ex-siRNAs is not random. Another characteristic of this class is that most of these genes are highly expressed, as denoted by the sequencing of high numbers of ESTs. Further experiments are needed to identify what other features, besides high expression, are required for the mRNAs to enter this pathway and exactly how class 3 ex-siRNAs are generated. Finally, class 4 is a tiny group of ex-siRNAs that derive from only ﬁve exons. These are the only ex-siRNAs that are down-regulated in dcl1 but not in dcl2 . Only one of these exons shows a reduced accumulation in the double dcl mutant, although two others are just below the 4-fold threshold in the dcl1 /dcl2 strain (Supplementary Table S3). We cannot rule out the possibility that some of the ex-siRNAs of this class is only an artefact of the sequencing, since it is diﬃcult to explain why the double dcl mutant is diﬀerent from dcl1 . However, we can point out that one of the exons included in this class (ID: 27711) encodes a conserved protein that in other fungi, such as yeast, co-localize with other proteins in sites of polarized growth (tip of the hypha) (19,20). This, together with the fact that other exons code for proteins involved in mitochondria metabolism and ribosome function may help to understand the phenotype of the dcl1 mutant, that is, abnormal hyphal morphology and a decrease in the growth rate. The biogenesis and function of the class 2 ex-siRNAs, the largest group of ex-siRNAs we have identiﬁed in M. circinelloides, somewhat resemble that of endogenous siRNAs in animals and plants. However, there are clear diﬀerences between these ex-siRNAs and previously described esRNAs. The most similar group is the en- dogenous siRNAs in C. elegans, which regulate the expres- sion of protein coding genes. However, these are mainly generated directly by the RdRP activity without the par- ticipation of a Dicer enzyme (21–23). The 24-mer hetero- chromatin siRNAs in plants are produced by Dicer from an RdRP generated dsRNA, but these siRNAs predom- inantly target transposons and repeat elements (24). They are therefore similar to the Mucor esRNAs mapping to transposons. Endo-siRNAs found in Drosophila and mouse oocytes are similar to class 2 ex-siRNAs because some of these target protein coding genes, but this class of esRNAs is produced from complementary transcripts without RdRP activity (4). The class 2 ex-siRNA pathway involves elements from all these diﬀerent pathways. Thus, dsRNA is generated by Mucor RdRP, similarly to plants (heterochromatin-siRNA), the dsRNA is cleaved by Dicer, as occurs in plants (heterochromatin-siRNAs) and higher animals (endo-siRNA) and the generated ex-siRNAs target protein coding genes in cis, which is similar to animals (endo-siRNA and secondary siRNAs). Therefore, the ex-siRNA pathway in Mucor uses all these known elements but in a new and unique combination. A new class of esRNA, qiRNAs, was recently reported to be involved in DNA-damage response in another ﬁla- mentous fungus, Neurospora crassa (25). Here, we identiﬁed another class of esRNAs that potentially regulate up to 276 mRNAs, suggesting that this layer of regulation is extensive in Mucor and potentially in other fungi. Indeed, the dcl mutants are aﬀected in hyphal morphology and colony growth (dcl1 ) (10) and in the production of asexual spores (dcl2 ) (8). We identiﬁed ex-siRNAs in fungi grown in optimal conditions but