conserved regulatory functions may shed light on disease mechanisms [6,42]. Thus, we considered it a timely moment to pause and compile an overview of sequence miRNA relationships in invertebrate genetic models. Given the expanded C. elegans, D. melanogaster and human miRNA identifications and the importance of rapidly identifying potential functional redundancies within and between species, we probed miRNA sequence relationships to compile a current list of mature miRNA sequence families within the C. elegans and D. melanogaster genomes, and we identified their human counterparts. Our analysis presents an overview that significantly expands the memberships of described sequence-related groups within, and between, species. We highlight new sequences conserved between species pairs or among nematodes, flies and humans. This compilation of sequence relationships should facilitate studies on miRNA evolution and conserved function that will contribute to enhanced understanding of complex miRNA regulatory networks and their biological activities. Results Recent reports have markedly expanded the numbers of identified miRNAs expressed in C. elegans, D. melanogaster, and humans [10,11,25,47,51–69]. Given the tremendous potential of invertebrate genetics to address in vivo function of conserved miRNAs, the availability of genetic knockouts of most of the 139 reported C. elegans miRNA genes, and our interest in evaluating miRNA contributions to cellular robustness and mechanisms of aging, we sought to generate a current overview of miRNA sequence families identifiable by comparisons among these species. We compared all reported C. elegans 139, D. melanogaster 152 and human 733 mature miRNA sequences available in the miRNA database miRBase 10.1 [43–46] using the ClustalW algorithm [70] to identify intra-species and inter-species sequence similarities. We classified miRNAs as sequence-related based on current under- standing of functional miRNA-target interactions, which may occur via either of two sequence relationships: 1) perfect complementarity of miRNA 59 end sequences, especially at nucleotide positions 1,2–8 referred to as the ‘‘seed’’ region, and 2) high level complementarity across the length of the miRNA (.70% identity overall) that can have less precise pairing in the seed region. 59 end seed region search criteria 59 end sequences are critical for miRNA function [2,41,71–73] and the seed region is thought to contribute to target recognition by perfect (or near perfect) complementary binding to the mRNA target site. The requirement for uninterrupted homology may render the miRNA-mRNA seed region under considerable selective pressure. Indeed, seed regions are highly conserved in mRNAs across species [32,41,74]. We therefore searched for 59 end seed matches that featured at least 7 continuous nucleotides of homology within the first 10 nt of the miRNA, a modestly relaxed criteria chosen to provide confidence that most potential seed region sequence relationships would be identified by this search. We did not allow interruptions (mismatches or gaps) within the first 10 nt except base changes that would permit G..U pairing, because G..U base pairing in the seed region has been documented to be possible in vivo under conditions of efficient miRNA target regulation [73]. Homology over miRNA length criteria Because some miRNAs have less stringent seed region pairing and instead exhibit homology to target transcripts over their lengths [41,71], we also compiled a list of related miRNAs using MicroRNAs of Worms and Flies the criteria of full-span homology. To establish a reasonable homology cut-off value, we examined previously identified miRNA families and determined that a score of $70% sequence similarity over length should identify most, if not all, miRNA homologs known from published target site models and miRNA groups. Because the current mechanism of action of miRNAs has been inferred from only very few validated miRNA-gene targets, we also elected to list miRNAs that exhibit 60–69.9% identity in supporting information. According to current understanding, the potential functional significance of the 60–69.9% similar miRNAs is of higher probability if the homology in the seed region is high. Overview: Sequence relationships that expand miRNA family lists in nematodes, flies and humans We performed alignments by both 59 end seed matching (nt 1–10) and by analysis of homology across complete mature miRNA sequences, comparing individual C. elegans miRNA sequences against all known C. elegans, D. melanogaster and human miRNAs, and individual D. melanogaster miRNAs against D. melanogaster and human miRNAs (Tables 1–6). Our analysis greatly expands the documentation of miRNA sequence family members. For example, our combined list of C. elegans miRNA sequence relationships identified 211 sequence relationships, placing 84 sequence-related miRNAs into 20 family groups (Table 1), whereas previous reports on C. elegans miRNAs [43,51,63,64] identified 110 sequence relationships between 70 miRNAs. Similarly, our combined searches for D. melanogaster miRNAs detected 126 sequence relationships including 70 sequence-related miRNAs in 24 family groups (Table 2), a considerable expansion of the previously reported 53 sequence relationships between 31 miRNAs [25,43,52,57,60,65,75]. Our work increases the number of C. elegans miRNAs with identified human counterparts to 76 (Table 4) and D. melanogaster miRNAs with identified human counterparts to 83 (Table 5) [25,43,51,52,57,60,63–65,75,76]. Significantly, we associated as many as 133 human miRNA sequences with sequence-related worm and/or fly miRNAs (Tables 4–6 and Figure S1). Below we highlight some details of C. elegans and D. melanogaster miRNA family searches and then discuss the invertebrate miRNAs that have clear human counterparts. C. elegans miRNA families C. elegans miRNA families defined by searches for homology in 59 end sequences. We searched for 59 end sequence alignments that included at least 7 nucleotides of continuous similarity within nt 1–10 of the mature miRNA, with no allowed gaps and only G..U mismatches permitted. By these criteria, we identified 81 C. elegans miRNAs that can be placed into 19 different families (Table 1, Dataset S1). We observed that 59 homologies were mainly located from nucleotides 2 to 8, consistent with conserved sequence present in the seed region (Figure S2). Moreover, related miRNAs sharing longer nucleotide homologies at the 59 end tend to be more similar at the 39 end (and therefore over their full lengths) as compared to miRNAs with 59 homologous regions of only 7 or 8 nucleotides. C. elegans miRNA families defined by searches for homology over their lengths. We also compiled a list of miRNA families by requiring homology over the entire miRNA length. We grouped 45 of the 139 C. elegans mature miRNAs into 15 different families based on $70% identity over mature sequence length (Table 1, Dataset S2). Consistent with current reports in the field, the highest similarity occurs predominantly at the 59 end in full-length sequence alignments. Two homology search criteria generate a C. elegans miRNA family list with substantial, but not complete, PLoS ONE | www.plosone.org 2 July 2008 | Volume 3 | Issue 7 | e2818