Gunaratne et al. BMC Genomics 2011, 12:277 http://www.biomedcentral.com/1471-2164/12/277 Table 3 Song-regulated targets of tgu-miR-2954-3p Ensembl ID Gene Symbol EST Gene Name Page 8 of 14 ENSTGUG00000001349 ELAVL2 ENSTGUG00000001404 LINGO2 ENSTGUG00000003073 TLK2 ENSTGUG00000008207 BTG1 ENSTGUG00000008540 CHD2 CK313262 ELAV-like protein 2 (Hu-antigen B)(HuB)(ELAV-like neuronal protein 1)(Nervous system-specific RNA-binding protein Hel-N1) DV957508 Leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor-interacting protein 2 Precursor (Leucine-rich repeat neuronal protein 6C)(Leucine-rich repeat neuronal protein 3) CK305975 Serine/threonine-protein kinase tousled-like 2 (EC 2.7.11.1)(Tousled-like kinase 2)(PKU-alpha) CK303273 Protein BTG1 (B-cell translocation gene 1 protein) DV958991 Chromodomain-helicase-DNA-binding protein 2 (CHD-2)(EC 3.6.1.)(ATP-dependent helicase CHD2) ENSTGUG00000010181 XP_002196848.1 CK304764 crk-like protein (v-crk avian sarcoma virus CT10 oncogene homolog-like) (CRKL) ENSTGUG00000010364 NEGR1 ENSTGUG00000011700 HMGB1 DV954047 Neuronal growth regulator 1 Precursor CK314519 High mobility group protein B1 (High mobility group protein 1)(HMG-1) We used TargetScan to find binding sites of tgu-miR-2954-3p on eight chicken genes and here are listed the information of their homologous genes in the zebra finch genome including Ensembl IDs, Gene Symbols, EST (Accession numbers of song-regulated EST identified in the previous microarray study) and Gene Names (or aliases in parenthesis). assemblies and may give rise to miRNAs that have not yet been described elsewhere due to low copy number, restricted tissue distribution or other factors. The remaining novel miRNAs, 34 in number, may be unique to the zebra finch or the songbird lineage. Few studies have attempted de novo identification of miRNAs from the brain [18] and ours is the first to report direct sequencing of songbird brain miRNAs. A previous study did identify precursor sequences for five conserved miR- NAs in the developing zebra finch brain [40]. Also, in parallel with our own Illumina analysis, Li and her col- leagues used 454 sequencing to identify miRNAs in the brain and liver of adult zebra finches. These different sets of annotations are compared and collated in a sup- plement to the analysis of the zebra finch genome assembly [33]. By comparing birds hearing novel song playbacks or silence, we found evidence for experience-dependent fluctuations in large numbers of miRNAs in the auditory forebrain. We performed three separate pairwise com- parisons by Illumina, where all aspects of the experi- mental conditions were carefully counterbalanced between the two groups in each comparison. The three comparisons were not direct replications of each other, as each had a different sex ratio. Our reasons for varying the sex ratio were partly pragmatic (limited numbers of birds of the same sex that could be removed from our aviary) and partly analytical (males and females have dif- ferent behavioral responses to songs). Some of the dif- ferences between the three sets of results may reflect real biological differences in the responses of males and females. Indeed, our Northern analysis of the tgu-miR- 2954-3p confirms a sex difference in expression of this Z-linked miRNA gene. This is especially intriguing because we also obtained TaqMan evidence for both sense and antisense transcripts of this miRNA. One can imagine scenarios where different ratios of sense and antisense transcription occur in males (two copies of the gene) and females (one copy of the gene) with different consequences on the transcriptional networks affected by song exposure in the two sexes. Ignoring the potential effects of sex, we identified five miRNAs that showed significant and consistent changes in response to song across all three Illumina compari- sons. Three miRNAs consistently decreased after song (tgu-miR-92, tgu-miR-124, tgu-miR-129-5p) and two increased (tgu-miR-25, tgu-miR-192). The down-regu- lated miRNAs are at much higher abundance (> 1000 reads in each run) and perhaps for this reason we were more successful at detecting them and replicating their song regulation by TaqMan assay in subsequent experi- ments with additional groups of birds. The most abun- dant miRNA in our regulated set, tgu-miR-124, consistently met the statistical test for significant down- regulation by song, in each of six separate experiments (three Illumina comparisons, two TaqMan analyses in Additional File 1, Table S4, and the TaqMan compari- son of song vs. SEN in Additional File 2, Figure 3). In studies in other species, miR-124 has been linked to brain plasticity and development in several contexts. Chronic cocaine administration results in down-regula- tion of miR-124 in the rodent mesolimbic dopaminergic system [41]. In the developing chick neural tube, miR- 124a is a component of a regulatory network that con- trols the transition between neural progenitors and post-mitotic neurons [42]. miR-124 also regulates adult neurogenesis, and its overexpression promotes neuronal differentiation [42,43] and neurite outgrowth [44]. Intri- guingly, in songbirds neurogenesis continues in the fore- brain throughout adulthood, from a population of precursor cells that line the walls of the lateral ventricles and have the characteristics of neural stem cells [45-47].