Song exposure regulates known and novel microRNAs in the zebra finch auditory forebrain

Abstract

Background: In an important model for neuroscience, songbirds learn to discriminate songs they hear during tape-recorded playbacks, as demonstrated by song-specific habituation of both behavioral and neurogenomic responses in the auditory forebrain. We hypothesized that microRNAs (miRNAs or miRs) may participate in the changing pattern of gene expression induced by song exposure. To test this, we used massively parallel Illumina sequencing to analyse small RNAs from auditory forebrain of adult zebra finches exposed to tape-recorded birdsong or silence.

Results: In the auditory forebrain, we identified 121 known miRNAs conserved in other vertebrates. We also identified 34 novel miRNAs that do not align to human or chicken genomes. Five conserved miRNAs showed significant and consistent changes in copy number after song exposure across three biological replications of the song-silence comparison, with two increasing (tgu-miR-25, tgu-miR-192) and three decreasing (tgu-miR-92, tgu-miR-124, tgu-miR-129-5p). We also detected a locus on the Z sex chromosome that produces three different novel miRNAs, with supporting evidence from Northern blot and TaqMan qPCR assays for differential expression in males and females and in response to song playbacks. One of these, tgu-miR-2954-3p, is predicted (by TargetScan) to regulate eight song-responsive mRNAs that all have functions in cellular proliferation and neuronal differentiation.

Conclusions: The experience of hearing another bird singing alters the profile of miRNAs in the auditory forebrain of zebra finches. The response involves both known conserved miRNAs and novel miRNAs described so far only in the zebra finch, including a novel sex-linked, song-responsive miRNA. These results indicate that miRNAs are likely to contribute to the unique behavioural biology of learned song communication in songbirds.

Figure 1

Pipeline with yields for analysis of putative novel miRNAs. 52 small RNA sequences did not align to miRBase reference sequences and were assessed for miRNA potential. 48 sequences passed the minimum criteria and were categorized into three groups according to strength of evidence (sequences are color-coded in Additional File 1, Table S3, as indicated). Seven (7) are confirmed novel miRNAs since they had all the characteristics of known miRNAs and in addition also had a less abundant miR* sequence that maps on the opposite side of the stem from the putative novel miRNA. These are labelled green in Additional File 1, Table S3. Twenty-one (21) putative novel miRNAs are highly confident (labelled blue) since they also shared characteristics of known miRNAs but no sequence was found aligning to the miR* region. Given that the miR and miR* sequences for most known miRNAs have vastly different copy numbers such that the miR* sequence is sometimes not found, the highly confident candidates are also highly likely to be genuine novel miRNAs, Twenty (20) candidates (labelled grey) had a subset of the characteristics of known miRNAs but not all and therefore were deemed potential candidates that require more evidence.

Figure 2

The genome locus for tgu-mir-2954 produces three different miRNAs. A. Alignments via the UCSC Genome Browser of the three detected miRNAs to the intron of the zebra finch XPA gene. B. Hairpin precursors for the three miRNAs. C. Northern blot analysis using an RNA probe complementary to novel miRNA tgu-miR-2954-3p.

Figure 3

Analysis of miRNAs produced at the tgu-mir-2954 locus. TaqMan and Illumina RNA-seq data generated from independent sets of birds (n = 6 in each data set) for expression from the tgu-mir-2954 locus. A) TaqMan results, where the relative gene expression of each individual bird (open circle) was obtained by using the 2^-ddCt method [98]; the relative gene expression of either Silence (white bar) or Song (gray bar) group was the mean of six individuals; the P value was calculated by paired t test since each song stimulated animal was explicitly paired with a silence control animal collected simultaneously. B) Read counts from the Illumina RNA-seq for miR-2954-3p and miR-2954-5p (also shown in the Additional File 1, Table S4).