Gunaratne et al. BMC Genomics 2011, 12:277 http://www.biomedcentral.com/1471-2164/12/277 non-song auditory stimuli [6,7,34,35]. To test for song- specificity of the miRNA response, we conducted a further TaqMan experiment assessing the levels of six miRNAs (tgu-miR-124, tgu-miR-92, tgu-miR-129-5p, and three miRNAs derived from the tgu-miR-2954 locus, next section), in birds who had heard either a normal song or a carefully matched non-song acoustic stimulus, “song enveloped noise” (SEN). SEN has the same amplitude envelope as the song from which it is derived but spectral content has been randomized so it does not sound like a song [34]. By TaqMan PCR, we confirmed that normal song induced a larger increase in zenk mRNA in these birds than did SEN (Additional File 2, Figure S3 panel D). In these same animals, nor- mal song, but not SEN, triggered a significant decrease in the levels of tgu-miR-124, tgu-mir-129-5p, tgu-miR- 92 and tgu-miR-2954-3p (Additional File 2, Figure S3 panels A-C, H). Thus we conclude that there is indeed a unique miRNA response in the auditory forebrain that is selective for song over non-song acoustic stimuli. A complex sex-linked miRNA locus in zebra finch and other birds The novel miRNA, tgu-mir-2954, that was detected most frequently in our Illumina assays maps to the sense strand of an intron in the XPA gene, on the Z chromosome (Figure 2A). The precursor hairpin con- tains reads from both arms, thus meeting our bioinfor- matic criteria for a confirmed miRNA (Figure 2B). By contrast to most known miRNAs, the numbers of reads from both 5’ and 3’ arms were found at similar copy numbers, suggesting that both arms may make func- tional mature miRNAs. BLAST analysis of the mir-2954 hairpin precursor sequence against the NCBI nr data- base identified a putative mature miRNA in chicken (gi| 145279910|emb|AM691163.1|), and BLAT analysis of a collection of transcripts from crocodile and 11 other bird species [36] detected mir-2954 transcripts in 2 non- passerine species (two hummingbirds) and 3 passerine species (the American crow, the pied flycatcher, and the golden collared manakin) (Additional File 2, Figure S2). There was no BLAT hit in the crocodile, the remaining 3 non-passerine birds (Emu, budgerigar, and ringneck dove), and 3 passerine species (collared flycatcher, blue tit and Eastern phoebe). The lack of a hit does not necessarily mean absence of the gene as these datasets represent incomplete transcriptomes derived by 454 sequencing [36]. These results clarify that the sequence is not unique to the zebra finch or passerines, but may nevertheless have a restricted distribution within birds. To validate the existence of these two miRNAs in zebra finch, we performed TaqMan analyses for both, using their reverse complements as controls. Interest- ingly, we got significant expression values not only for Page 5 of 14 the predicted miRNAs but also for one of the reverse- complement miRNAs (tgu-miR-2954R-5p) although no significant song regulation for miR-2954R-5p was found (Additional File 2, Figure S3 panels I-J). With respect to the XPA gene within which this locus is embedded (Fig- ure 2A), these data suggest that precursor-miRNA-stem loops are produced from both the sense (same orienta- tion as XPA) and antisense strands. The stem loop pre- cursor processed by Drosha from the sense RNA (tgu- mir-2954) generates two active miRNAs from its both arms (tgu-miR-2954-3p and tgu-miR-2954-5p). The stem loop precursor processed by Drosha from the anti- sense RNA (tgu-mir-2954R) generates at least one active miRNA (tgu-miR-2954R-5p) from its 5’ end sequence. We carried out Northern analysis on tgu-miR-2954- 3p, which is the miRNA that has the highest number of read counts detected in our Illumina assays among the three miRNAs from the tgu-mir-2954 locus. A robust signal at ~22 nucleotides is evident in mixed-sex pools of RNA from birds hearing either song or silence, and a weaker signal is also detectable in two female-only pools of RNA (Figure 2C). Greater expression in males is con- sistent with the ZZ genotype of males and the lack of efficient sex chromosome dosage compensation in the zebra finch [37,38]. By TaqMan as well as by Illumina, we observed an apparent sex difference in the direction of the response of tgu-miR-2954-3p to song - up in males and down in females (Figure 3 and Additional File 1, Table S4). This suggests this locus may be under complex regulation, integrating information about sex, auditory or social experience and perhaps also other factors related to XPA gene expression. To gain insight into the potential functional role of tgu-miR-2954-3p in the response to song, we used a conservative strategy to predict gene targets that are both conserved in birds and responsive to song expo- sure in the zebra finch. Potential targets of miRNAs are described as mRNAs that have sequences that can undergo Watson-Crick base pairing with the 5’-seed (nucleotide 2-7) of the miRNA [39]. For target predic- tion we applied the TargetScan (5.1) algorithm using the chicken genome as an initial reference, and then con- firmed presence of the target sequence in the zebra finch. For evidence of song responsiveness, we used the data set of Dong et al. [14]. Eight genes met all these criteria (Table 3) and are thus both song-responsive and also subject to regulation by tgu-miR-2954-3p. These genes all have functions in control of cell proliferation or neurite outgrowth (see below). Discussion Here we show that a natural perceptual experience, hearing the sound of another bird singing, alters the