the same transcriptional orientation within relatively short distances can be considered as clustered. 42% of human mir genes appear in clusters of 2 or more within 3 Kb intervals [79]. Some C. elegans mir gene clusters have been previously described: mir-35-mir-41 (within a 796 bp region), mir-42-mir-44 (307 bp), mir- 54-mir-56 (403 bp), mir-229_mir-64-mir-66 (754 bp), mir-73-mir-74 (374 bp), and mir-241_mir-48 (,1.7 Kb) [61,63,80]. Genes within these groups exhibit similar expression patterns, indicating that they might be co-transcribed into polycistronic units. Based on these observations, we chose a potential clustering range of 2 Kb to evaluate relative mir gene distribution in the C. elegans genome (Figure 1A). Interestingly, 35/137 C. elegans mir genes cluster into a total of 13 groups by this criterion. Most of the clusters contain 2 mir genes, with clustered mir genes more abundant on chromo- somes II and X (the latter of which harbors a higher than average number of mirs overall (Figure S4A)). We checked whether clustered mirs are related in sequence and found that about half of the mir clusters contain mir genes that are homologous at the 59 end and/or over full length ($70%). If co-expressed, such genes might regulate common mRNAs by recognizing the same target sites. mirs in the remaining half of the clusters do not exhibit significant homology between them. If these mirs are co-expressed, they may target different mRNAs or might interact with the same target transcripts via multiple, distinct miRNA binding sites. We also looked at the distribution of mir genes in the D. melanogaster genome (Figure 1B). Consistent with previous reports [52,60], we determined that 60/152 Drosophila mir genes are clustered into 20 different regions 2 kb long. Clusters contain on average 3 mir genes with the longest cluster including 8 mir genes. Clustered mir genes are more abundant on chromosomes 2L and 2R, which also have a higher than average number of mirs overall (Figure S4B). ,38% of the clustered mir genes in the Drosophila genome have 59 and/or $70% full homologous sequences. D. melanogaster miRNA families Similar to our strategy for C. elegans miRNA analysis, we screened D. melanogaster miRNAs for 7 consecutive identical nucleotides at the 59 ends and classified 61 miRNAs into 19 families (Table 2, Dataset S4). Using the criteria of $70% overall identity, we highlight a total of 38 miRNAs that can be classified into 14 families (Table 2, Dataset S5). Overall, we identified 70 of the 152 Drosophila miRNAs as part of 24 sequence-related groups (Table 2). As is the case for C. elegans miRNAs, lists of related Drosophila miRNAs compiled by the 59 and $70% search criteria overlapped. However, 48 fly miRNAs are significantly similar at their 59 end but have ,70% overall identity with at least one of their sequence-related miRNAs (indicated in Dataset S4). Of these, 33 miRNAs have significant homology to other fly miRNAs only at their 59 end and thus are not listed in the $70% homology groups (Table 2, Dataset S5). Most of the fly $70% full length homologs exhibit blocks of $7 nt identity at the 59 end except the following 10: dme-miR-10, dme-miR-100, dme-miR-263a, dme- miR-263b, dme-miR-954, dme-miR-966, dme-miR-1009, dme- miR-1010, dme-miR-iab-4-3p and dme-miR-iab4as-3p. miRNAs conserved between C. elegans and D. melanogaster We next compiled an expanded list of sequence-related miRNAs common to nematodes and flies. We searched for both 59 end matches and for $70% homology over extended length between the 139 C. elegans and 152 D. melanogaster miRNAs using the criteria we described above for intra-species comparison. Overall, our sequence comparisons establish 64 novel worm/fly MicroRNAs of Worms and Flies miRNA relationships [25,43,51,52,57,60,63–65,75] and identify 87 miRNA families that now include 87 C. elegans and 65 D. melanogaster members (Table 3). 59 end homology searches detected 87 worm miRNAs related to 62 fly miRNAs (Table 3, Dataset S7), whereas $70% overall identity searches highlighted 31 worm miRNAs and 37 fly miRNAs in family relationships (Table 3, Dataset S8). Of the 87 59 related C. elegans miRNAs, 68 have a $7 nt block homology at the 59 end but weak full length identity (,70%) with at least one of their 59 fly miRNA relatives (indicated in Dataset S7). 59 of these have ,70% full length sequence similarity with all their 59 Drosophila relatives and thus these relationships are not present in our $70% homology lists in Dataset S8. 15 of the 87 C. elegans miRNAs with 59 identities in flies have significant extended homology over their full length ($70%) with all their Drosophila counterparts. Most of the C. elegans_Drosophila $70% miRNA homologs have $7 nt identity at the 59 end except cel-miR- 239a_dme-miR-12, cel-miR-252_dme-miR-252 and cel-miR- 250_dme-miR-1007. miRNAs conserved between C. elegans and H. sapiens We also searched for both 59 end identities and for homologous ($70%) extended sequence between C. elegans (139) and human (733) miRNAs using the criteria we described above. Overall, our sequence comparisons establish 141 novel nematode_human relationships [43,51,63,64,76] and identify 76 miRNA families that now include 76 C. elegans and 102 human members (Table 4). 76 worm miRNAs exhibit significant homologies to the 59 ends of 98 human miRNAs (Table 4, Dataset S10), whereas 22 nematode miRNAs are $70% homologous over their full length to 46 human miRNAs (Table 4, Dataset S11). 69 of the 76 59 related C. elegans miRNAs have ,70% extended homology with at least one of their 59 human counterparts (shown in Dataset S10), and 54 are weakly similar (,70%) with human miRNAs outside their 59 end sequences. 7 of the above 76 C. elegans miRNAs have significant 59 and overall ($70%) homology with all their 59 related sequences in humans. In our set of C. elegans_human $70% homologs, the following do not have $7 nucleotides of continuous similarity at the 59 end: cel-miR-57 with hsa-miR-99a and hsa-miR-100; cel- miR-236 with hsa-miR-141 and hsa-miR-200a; and cel-miR-266 with hsa-miR-25*, hsa-miR-301a and hsa-miR-301b. miRNAs conserved between D. melanogaster and H. sapiens Looking for 59 end and $70% overall sequence similarities between D. melanogaster (152) and human (733) miRNAs, we detected 149 novel sequence relationships previous reported in [25,43,52,57,60,65,75,76] expanding family groups to 83 defined by 83 Drosophila miRNAs and 121 human miRNAs (Table 5). Specifically, 82 Drosophila miRNAs show significant 59 sequence identity to 117 human miRNAs (Table 5, Dataset S13), and 40 fly miRNAs are $70% homologous over full length to 56 human miRNAs (Table 5, Dataset S14). 67 of the above 82 Drosophila miRNAs are ,70% identical to the full sequences of some of their 59-related human miRNAs (identified in Dataset S13)—45 are weakly similar (,70%) to all their 59 related human sequences outside the 59 region. The remaining 15 of the 82 Drosophila miRNAs have $70% overall homology in addition to 59 relation to all their 59 human counterparts. 8 of the 40 Drosophila miRNAs with $70% homologous sequences in humans show extensive overall similarity with 59 mismatches: dme-miR-8 with hsa-miR- 141 and hsa-miR-200a, dme-miR-10 with hsa-miR-100 and hsa- miR-99a, dme-miR-100 with hsa-miR-10a and hsa-miR-10b, dme-miR-125 with hsa-miR-10a and hsa-miR-10b, dme-miR263a PLoS ONE | www.plosone.org 17 July 2008 | Volume 3 | Issue 7 | e2818