5536 Nucleic Acids Research, 2010, Vol. 38, No. 16 diﬀerent classes of antisense esRNAs, 21 and 25 nt long (9). Null dcl2 mutants (dcl2 ) show a signiﬁcant reduction in the production of asexual spores, which suggests a role for dcl2 in the control of vegetative development (8). Similarly, mutants aﬀected in the dcl1 gene (dcl1 ) show defects in vegetative growth and hyphal morphology, even though this gene is not required for eﬃcient gene silencing triggered by transgenes (10). In addition, two genes coding for RNA-dependent RNA polymerases (RdRPs) are involved in the silencing mechanism in M. circinelloides. rdrp1 is required for sense transgene-induced silencing to generate dsRNA molecules but the role of rdrp2 is not understood (S.C., S.T-M., R.M.R-V., our unpublished results). Relatively little is known about the physiological roles of the fungal RNA-silencing pathways (7). Availability of M. circinelloides mutants aﬀected in the silencing mechan- ism and the recent completion of its genome sequencing have provided the genetic and molecular tools required to investigate how esRNAs regulate gene expression in this basal fungus. We analyse here the esRNA content of M. circinelloides by deep sequencing of short RNAs (18–25 nt) in the wild type and several mutant strains and identify a new class of esRNAs that target mRNAs of protein-coding genes from which they were produced. MATERIALS AND METHODS Strains and growth conditions The leucine auxotroph R7B, derived from M. circinelloides f. sp. lusitanicus CBS 277.49 (syn. Mucor racemosus ATCC 1216b), was used as the wild-type strain. Strain MU406 is a dcl1 mutant derived from MU402, a uracil auxotroph derivative of R7B (8). Strain MU410 is a dcl2 mutant derived from MU402 (10). Strain MU411 is a double dcl1/dcl2 mutant derived from MU410 (10). Strains MU419 and MU420 correspond to rdrp1 and rdrp2 mutants derived from MU402, respectively (S.C., S.T-M., R.M.R-V., our unpublished results). Cultures were grown at 26 C in complete YPG medium as described previously (10). The pH was adjusted to 4.5 for mycelial growth. RNA analysis Short RNA samples were extracted from mycelia grown for 48 h on YPG plates using the miRVana kit (Ambion), following the instructions of the supplier. cDNA libraries Table 1. Sequencing of short RNAs in diﬀerent Mucor strains of short RNAs were generated and sequenced as described previously (11). Low and high molecular weight RNA was extracted from frozen mycelia and analysed by northern blot as described previously (9). Sequence analysis Raw sRNA reads were processed by removing adaptor sequences with exact matches to the ﬁrst eight bases of the 3’-adaptor. Annotated exon regions were extracted from the M. circinelloides genome (v 1.0) along with annotated transposons and repeat regions. All remaining segments of the M. circinelloides genome were used as the intergenic set for this analysis. sRNAs were mapped to exons, transposons/repeats and intergenic regions using PatMaN (12) and distinct sRNA producing loci were predicted in intergenic regions using a previously published method (13). Any locus containing fewer than ﬁve reads was discarded for the initial locus analysis. miRNA prediction was carried out both on intergenic regions and the full genome using a modiﬁed version of the miRCat pipeline described previously (14). In order to correct for variation in sRNA sample sizes and allow us to perform cross-sample expression analysis, sRNA counts for each of the intergenic, exonic and trans- poson loci were converted into reads per million genome matching reads. sRNA loci were said to be down- regulated in a given sample if the normalized locus abun- dance showed at least a 4-fold decrease in comparison to the wild-type sample. To increase the stringency of the analysis and avoid lowly expressed regions, any loci with a normalized abundance count of less than 50 in the wild type were excluded from the analysis. RESULTS AND DISCUSSION Short RNAs are primarily generated by DCL2 in Mucor Two dicer-like genes (dcl1 and dcl2) have been identiﬁed in Mucor and a possible function has been hypothesized for dcl2 gene (8). In order to identify bona ﬁde esRNAs we generated cDNA libraries of short RNAs from wild-type (dcl1/dcl2), single mutant (dcl1 /dcl2 or dcl1/dcl2 ) and double mutant (dcl1 /dcl2 ) strains (8 and 10) and sequenced them on the Solexa platform (Table 1; GEO accession number is GSE18958). Non-redundant reads were mapped to the genome, whose sequence was avail- able from the JGI (http://genome.jgi-psf.org/Mucci1/ Mucci1.home.html). In fact, this is the ﬁrst global analysis reported from the Mucor sequencing project. Total reads After adaptor removal (total) After adaptor removal (unique) Mapping to genome (total) Mapping to genome (unique) WT 6 421 725 5 701 786 1 362 728 4 317 874 777 409 dcl1 5 326 957 4 604 676 1 357 556 3 619 529 808 650 dcl2 6 155 439 5 049 070 1 354 177 3 912 401 890 709 dcl1 /2 10 899 332 9 303 440 3 027 587 7 094 985 2 121 808 rdrp 11 088 786 10 178 115 1 929 224 8 347 666 1 272 679 rdrp 10 846 130 9 877 133 2 654 222 7 880 896 1 512 498 The table shows the read numbers of diﬀerent categories for each strain we have analysed.