Gunaratne et al. BMC Genomics 2011, 12:277 http://www.biomedcentral.com/1471-2164/12/277 may be particularly important for the evolution of brain and behavior. Many miRNAs are expressed in the brain [16], often in different patterns in different species [17-19]. Brain miRNAs undergo dramatic changes in expression during development [20-22] and aging [23] and have been functionally implicated in neurological disease [24]. They may also function in the normal phy- siological operation of the nervous system as suggested by evidence for involvement of miR-132 and miR-219 in circadian clock regulation [25] and miR-134 in control of dendritic translation [26,27]. Here we apply massively parallel Illumina sequencing to probe the involvement of miRNAs in the processing of song experience in the zebra finch auditory forebrain. We begin by identifying 155 different miRNA sequences and the genomic loci of their precursor sequences in the zebra finch genome, including 34 miRNA genes that have not been detected in the genomes of other species. We then ask whether the miRNA content changes after song exposure and find robust evidence of miRNA responses to song playbacks. We also assess correlations between expression changes of a novel miRNA and its predicted target mRNAs during song habituation. The results indicate an active role for miRNAs in the neural processing of a natural perceptual experience - hearing the sound of another bird singing. Results The miRNAs of the zebra finch auditory forebrain We carried out Illumina small RNA sequencing (RNA- seq) on the small RNA (~18-30 nucleotides) fraction of total RNA isolated from adult zebra finch auditory fore- brain. Ultimately, we performed 6 Illumina runs on 6 dif- ferent RNA samples, to assess the effects of song exposure (next section). First we describe the overall small RNA profile obtained by combining the results of all the runs, representing 36 adult zebra finches (equal numbers of males and females). A total of 20 million reads were obtained (Table 1) and aligned to reference miRNA sequences from other species (miRBase version 13.0). Overall we identified 107 non-redundant miRNAs representing 52% of sequences that have been previously identified in chicken, rodent and human. The remaining sequences mapping to the piRNA database were denoted as piRNA reads (~30%) (Additional File 1, Table S1). Table 1 Summary statistics for the read alignments Page 2 of 14 Reads that did not align to known RNAs were assessed for miRNA potential through a novel miRNA discovery pipeline described by Creighton et al.[28] which tests for properties that are characteristic of known miRNAs. These properties include the following: 1) The mature sequence must map to the stem region of the hairpin sequence of the putative precursor extracted from the zebra finch genome. 2) The mature miRNA sequence must map to the precursor such that it can be processed following the Drosha processing rules [29]. All novel miRNA candidates that map to the loop region and/or lack appropriate Drosha processing sites are failed. 3) Known miRNAs have stable 5’-ends that vary at the most by +/- 1 nucleotide. 4) By contrast the 3’-ends of miRNAs are highly heterogeneous in length due to imprecise Dicer processing [29,30] and exhibit non-templated nucleotide sequence changes due to RNA editing [29-31]. 5) Consequently, the putative precursor must give a strong signal of sequence align- ments in a tight area of 18-25 nucleotides. Small RNA sequences that are distributed fairly evenly along the entire length of the precursor are rejected since they likely represent degraded products of a large RNA. The candidates that also demonstrate the presence of the miRNA star sequence (miR*) mapping on the opposite side of the mature miRNA and occurring at a lower abundance in the deep sequencing data are considered to be confirmed novel miRNAs in zebra finch. Using this pipeline (Figure 1) we discovered 48 putative novel miRNAs that map on the zebra finch genome to a stem loop structure that folds with a minimum free energy of < -20 kcal/mol [32]. The complete analysis and mapping information for all the novel miRNA candidates is given in Additional File 1, Tables S2 and S3. All novel miRNA candidates were mapped to genomic loci in the zebra finch genome assembly [33], and also to human and chicken genomes using the BLAT func- tion of the UCSC Genome Browser (Additional File 1, Table S3). In the zebra finch genome, the loci include both annotated exons and introns as well as unanno- tated intergenic regions. Thirty-four (34) novel micro- RNAs uncovered from zebra finch are not presently detected in the human or chicken genome assemblies. Eleven (11) map to genome positions in chicken, and six to positions in the human (with three of these found in Male silence Male song Female silence Female song Mix silence Total Reads Total Usable Reads Reads aligning with known miRNA Total Fraction 2,704,778 1,179,330 401,934 34% 2,056,391 1,155,168 209,944 18% 3,173,108 2,244,376 1,638,528 73% 3,546,038 2,498,648 1,755,748 70% 3,962,050 2,249,188 1,348,109 60% Six different pools of auditory forebrain were analyzed independently by Illumina small RNA sequencing, as described in the text. Mix song 4,738,528 2,950,398 2,113,006 72%