of complex higher organisms, or possibly remaining to be discovered in human genomes. 6 nematode miRNAs have $70% homologous counterparts in humans but currently lack identifiable family members in Drosophila. 28 C. elegans miRNAs have $70% sequence similarity with other C. elegans miRNAs but were not found in either fly or human genomes. In a similar manner, we inspected the conservation of D. melanogaster miRNAs in nematodes, flies and humans. 54 D. melanogaster miRNAs have homologous sequences both in nema- todes and humans (Figure 2). Searches with 59 end sequences identified 54 D. melanogaster miRNAs with 59-related sequences in both nematodes and humans, 9 in nematodes and 29 in humans. 11 D. melanogaster miRNAs have 59 related sequences only in flies and are not present or remain unidentified in nematodes and humans. Considering $70% identity over the entire length, 21 D. melanogaster miRNAs have $70% homology counterparts in both nematodes and humans, 16 in nematodes and 19 in humans. 15 D. melanogaster miRNAs have $70% similarity to only other fly miRNAs. Overall, analysis of most recent miRBase release data highlights significant conservation of many miRNAs, supporting that analysis of their biological activities in invertebrate models will shed insight into functions relevant to human biology. Discussion An overview of inter- and intra-species relationships among miRNAs miRBase release 10.1 (December 2007) identifies 733 human, 139 C. elegans and 152 D. melanogaster mature miRNAs [43–46]. This list of annotated miRNAs, compiled predominantly from large-scale sequencing studies, has grown at an impressive rate in the recent past–for example, the list of human miRNAs has increased by over 500 sequences during the last 3 years. Although miRNA identification efforts are unlikely to yet be complete, current documented miRNAs most likely represent abundant species processed from typical hairpin structures. The field now faces the challenge of determining the biological activities of these miRNAs. Recently, extensive collections of C. elegans mir mutants have been generated [48], defining an opportune moment at which to evaluate sequence-related families and conserved functions. In this paper, we present a comprehensive classification of all the miRBase 10.1 miRNA sequences annotated in C. elegans, D. melanogaster and humans into sequence-related groups to identify miRNAs with possible redundant functions in the same species and those with potentially conserved functions across species. This compilation, which takes into account the two ways in which functionally related mature miRNAs can be similar (either 59 end seed homology or homology over length), is based on mature miRNA sequences rather than precursor gene sequence and adds to the considerable numbers of documented sequence-related family members [25,43,51,52,57,60,63–65,75,76], providing de- tails of sequence relationships. Intraspecies analysis: many invertebrate miRNAs have potential for functional redundancy Looking within individual species, we find that ,60% (84/139) C. elegans miRNAs and ,46% (70/152) D. melanogaster miRNAs share significant homology with other miRNAs encoded by their respective genomes. The potential for functional redundancy of miRNAs is clearly considerable within these species. The importance of evaluating sequence-related miRNAs during functional analysis has been elegantly exemplified by work on the MicroRNAs of Worms and Flies C. elegans let-7 miRNA family. Sequence-related miR-48, miR-84 and miR-241 work together to regulate developmental timing by redundant complementarity to binding sites in the 39 UTR of hbl-1 [40]. mir-48, mir-84 and mir-241 single mutants are seemingly wild type at 20uC. However, double and triple combinations of mir-48, mir-84 and mir-241 mutations cause developmental defects, revealing biological roles for these family members and stressing the importance of the analysis of multiple homologous miRNAs during functional studies. Of course, sequence-related miRNAs might be expressed in different tissues or at different times in development, and therefore might be excluded from performing similar functions with common targets. Still, the extensive sequence relationships that we document underscore that potential co-expression of sequence-related miRNAs will be a significant factor in evaluation of genetic disruptions as well as in commonly executed over-expression studies. Information on the expression patterns of sequence-related miRNAs will be important to careful interpretation of experimental outcomes. The extent of conservation of miRNA sequences from invertebrates to humans is striking Another theme that our analysis underscores is the substantial conservation of miRNA sequences across species. ,62% C. elegans miRNAs are related to Drosophila miRNAs (87/139), ,55% C. elegans miRNAs are related to human miRNAs (76/139), and ,55% Drosophila miRNAs are related to human miRNAs (83/ 152). Over half of the C. elegans miRNAs share sequence homology with miRNAs expressed in both flies and humans (73/139), and this number should increase with an increase in reported miRNAs. The extensive conservation across species suggests that this group of miRNAs contributes important functions in biology and that experiments in one species may well inform on the biology of another. Indeed, cross-species analyses of let-7 miRNA function has already provided useful leads for addressing human disease regulation. let-7 represses C. elegans RAS ortholog let-60 [81], as well as the human RAS oncogene transcript [42]. Recently these findings have been extended to demonstrate that let-7 expression reduces tumor growth in mouse lung tumor models [82]. Taking stock in a dynamic field miRNA discovery is an highly active research area. Here we report 133 human miRNAs with related sequences encoded by the C. elegans and/or D. melanogaster genomes. The majority of cataloged human miRNAs have unknown functions. Gene knock-outs, chemically modified antisense oligonucleotides, decoy miRNA targets (miRNA sponges) and over-expression studies are currently being used to evaluate loss-of-function of miRNAs [48,83–86]. Functional investigation of sequence-related miRNAs from C. elegans and D. melanogaster in a whole-organism context will most certainly provide insight into miRNA roles in specific mechanisms relevant to normal development as well as disease. The numerous sequence relationships identified to date will help focus research on abundantly expressed, conserved miRNAs while additional miRNA discovery continues to expand known miRNA families. Methods miRNA sequences and criteria for family grouping Mature miRNA sequences in C. elegans, D. melanogaster and H. sapiens were retrieved from the miRNA registry release 10.1 (December 2007) in miRBase [43–46]. miRNAs in C. elegans, D. melanogaster, C. elegans-D. melanogaster, C. elegans-H. sapiens and D. melanogaster-H. sapiens were classified into homology groups based PLoS ONE | www.plosone.org 20 July 2008 | Volume 3 | Issue 7 | e2818