D136 Nucleic Acids Research, 2006, Vol. 34, Database issue Table 1. Statistics concerning the miRNA genes in the miRNAMap miRBase (release 6.0) (3) Putative miRNA genes Subtotal Human Known 131 131 Putative 117 464 581 Mouse Known 196 196 Putative 54 395 449 Rat Known 117 117 Putative 86 274 360 The amounts of the overlapping miRNAs between the putative miRNAs and miRBase known miRNAs in human, mouse, rat and dog are 122, 97, 44 and 1, respectively. Putative 0 336 336 23 18 15 3 Total Known 450 450 9 9 9 3 Table 2. Statistics concerning the known human miRNAs targets detected by miRanda considering different parameters miRanda MFE (kcal/mol) miRanda score >120 >140 >160 >180 < 12 < 15 < 20 < 25 four mammalian genomes are identiﬁed using RNAz (4), which is a tool for predicting non-coding structural RNAs based on comparative sequence analysis. A machine learning method was developed to locate accurately the positions of the mature miRNAs to determine the mature miRNAs that were located at the putative miRNA precursors. miRanda (1) was applied to identify energetically the most probable miRNA targets of the miRNAs against the conserved regions in 30-UTR of the genes in the four genomes, and thereby predict the target genes of the known miRNAs and the putative miRNAs. Finally, the rela- tionships between the miRNAs and the coding genes are elucidated based on the miRNA targets. The miRNAMap also yields the expression proﬁles of known miRNAs, cross-species comparisons, gene annotations and cross-links to other biological databases. Both textual and web interface are provided to facilitate the retrieval of data curated in the miRNAMap. The main contribution of this work is the computational establishment of mapping between the miRNA genes and the miRNA target genes, and the provision of effective anno- tations, including miRNA expression, cross-species compar- isons and the annotations of the miRNA target genes. A comparative sequence analysis yields a series of the putative miRNA precursors and the mature miRNA conserved in human, mouse, rat and dog genomes. Additionally, a variety of search functions and a graphical interface are designed and implemented to help researchers to investigate the microRNA roles in cell regulations. DATABASE STATISTICS The miRNAMap presently stores 2176 miRNAs in four mam- malian genomes—human, mouse, rat and dog—as presented in Table 1. The number of experimentally validated (known) miRNA genes in humans, mice, rats and dogs obtained from the miRBase (Release 6.0) (3) are 131, 196, 117 and 6, respec- tively. The number of putative miRNA genes in humans, mice and rats from the miRBase are 117, 54 and 86, respectively. The number of putative miRNA genes in humans, mice, rats and dogs identiﬁed in this database are 464, 395, 274 and 336, respectively. By sequence comparison between the putative miRNAs in the proposed resource and known miRNAs from miRBase (3), the amounts of putative miRNAs identical to known miRNAs in human, mouse, rat and dog genomes are 122, 97, 44, and 1, respectively. The minimum free energy (MFE) of the miRNA–target duplex was determined while predicting the miRNA target sites. The lower MFE values of the miRNAs and the target sites reveal the energetically more probable hybridizations between the miRNAs and the target genes. Table 2 presents statistics Putative 257 1469 1726 3 3 3 3 Dog Known 6 6 27 21 17 3 Table 3. miRNA genes categorized by genomic location Genomic Human locations Mouse Rat Dog Total Intergenic 396 (52%) 405 (62%) 359 (76%) 225 (64%) 1385 (62%) Intronic 308 (40%) 235 (36%) 112 (24%) 107 (31%) 762 (34%) Exonic 58 (8%) 12 (2%) 0 (0%) 18 (5%) 88 (4%) Subtotal 762 (100%) 652 (100%) 471 (100%) 350 (100%) 2235 100%) concerning the miRanda predicted known human miRNA targets, which are extracted from literature (Table S3). For instance, 27 known miRNA targets can be identiﬁed when the miRanda MFE threshold and miRanda score threshold are set to 12 kcal/mol and 120, respectively. However, these parameters are likely to grossly overpredict the number of miRNA targets per gene. Alternatively, the proposed database allows users to consider a set of parameters that is more strin- gent and gives less likely false positives. All the predictive miRNA/targets are determined and stored in the proposed miRNAMap if the MFE of miRNA/targets is < 12 kcal/ mol and the miRanda (1) score of miRNA/targets exceeds 120. For instance, when the MFE threshold is set to 20 kcal/mol and the miRanda score threshold is set to 120, the average number of target genes of each miRNA in humans, mice, rats and dogs are 1419, 1390, 293 and 760, respectively (Table S4). The average number of distinct miR- NAs that target each gene in humans, mice, rats and dogs are 131, 116, 80 and 15, respectively. Similarly, when the miRanda (1) score threshold is set to 160 and the miRanda MFE threshold is set to 12 kcal/mol, the average number of the target genes for each miRNA in human, mouse, rat and dog genomes are 183, 175, 40 and 273, respectively. The average number of the distinct miRNAs targeting each gene in human, mouse, rat and dog genomes are 16, 14, 10 and 6, respectively. Additionally, all of the miRNA genes were observed with reference to the annotations of the genes in the four genomes: e.g. 52, 40 and 8% of the human miRNA genes were present in the intergenic, intronic and exonic regions, respectively (Table 3). In summary, of all the miRNAs in the proposed