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Genome-wide annotation and analysis of zebra finch microRNA repertoire reveal sex-biased expression
- Guan-Zheng Luo†1,
- Markus Hafner†2,
- Zhimin Shi3,
- Miguel Brown2,
- Gui-Hai Feng1,
- Thomas Tuschl2,
- Xiu-Jie Wang1Email author and
- XiaoChing Li3Email author
© Luo et al.; licensee BioMed Central Ltd. 2012
- Received: 7 August 2012
- Accepted: 21 December 2012
- Published: 26 December 2012
MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally in a wide range of biological processes. The zebra finch (Taeniopygia guttata), an oscine songbird with characteristic learned vocal behavior, provides biologists a unique model system for studying vocal behavior, sexually dimorphic brain development and functions, and comparative genomics.
We deep sequenced small RNA libraries made from the brain, heart, liver, and muscle tissues of adult male and female zebra finches. By mapping the sequence reads to the zebra finch genome and to known miRNAs in miRBase, we annotated a total of 193 miRNAs. Among them, 29 (15%) are avian specific, including three novel zebra finch specific miRNAs. Many of the miRNAs exhibit sequence heterogeneity including length variations, untemplated terminal nucleotide additions, and internal substitution events occurring at the uridine nucleotide within a GGU motif. We also identified seven Z chromosome-encoded miRNAs. Among them, miR-2954, an avian specific miRNA, is expressed at significantly higher levels in males than in females in all tissues examined. Target prediction analysis reveals that miR-2954, but not other Z-linked miRNAs, preferentially targets Z chromosome-encoded genes, including several genes known to be expressed in a sexually dimorphic manner in the zebra finch brain.
Our genome-wide systematic analysis of mature sequences, genomic locations, evolutionary sequence conservation, and tissue expression profiles of the zebra finch miRNA repertoire provides a valuable resource to the research community. Our analysis also reveals a miRNA-mediated mechanism that potentially regulates sex-biased gene expression in avian species.
- Zebra finch
- Sequence variations
- Tissue-enriched miRNA expression
- Z chromosome
- Sex-biased miRNA expression
MicroRNAs (miRNAs) are short non-coding RNA molecules that regulate gene expression post-transcriptionally. miRNAs are transcribed by RNA polymerase II. Primary transcripts of miRNAs are cleaved in the nucleus by the nuclease Drosha to generate precursor miRNAs (pre-miRNAs) with a characteristic hairpin-like secondary structure[2–4]. The pre-miRNAs are then exported to the cytoplasm and further cleaved by the RNase III enzyme Dicer to release a 21–23 nt small RNA duplex from the stem region of the hairpins[2, 5–7]. In most cases, only one strand of the small RNA duplex is retained as mature miRNA, and the other strand, termed miRNA star (miRNA*), is degraded. In some cases, however, both strands from the same hairpin are retained as functional miRNAs, which are annotated as miRNA-5p and miRNA-3p, respectively, depending on which arm of the hairpin they are derived. Sequence variations, such as nucleotide deletion, insertion, untemplated terminal extension, and editing, have been observed for some miRNAs[10–13]. The majority of animal miRNAs function by binding to the 3′-untranslated regions (UTRs) of target mRNAs via sequence complementarity to induce mRNA degradation or repress protein translation[14–16]. miRNAs have been found in most eukaryotes and many of them are conserved through evolution. The recent application of deep sequencing technology has also revealed an increasing number of species-specific miRNAs[10, 17–21]. It is estimated that over 60% of human protein-coding genes might be regulated by miRNAs. By fine-tuning gene expression, miRNAs regulate many biological processes ranging from cell proliferation, cell fate specification, cell differentiation, apoptosis, animal development, metabolism, to various disease conditions[1, 16, 23].
The zebra finch (Taeniopygia guttata), an oscine songbird, provides a unique animal model for neurobiological research with its characteristic learned vocal behavior. Zebra finches use a discrete set of interconnected brain nuclei and pathways, commonly referred to as the song system, to control song behavior. Song behavior in zebra finches is sexually dimorphic: only male birds sing, and the song system is highly developed in males[26, 27]. For decades, zebra finches have been widely used to study vocal learning, neuronal replacement, and sexually dimorphic development of male and female brains[24, 28]. In recent years, large efforts have been devoted to developing genomic resources and extending research in zebra finches to molecular and genomics levels[29–35]. As part of this effort, we have systematically characterized miRNA expression in various tissues of adult female and male zebra finches using the Illumina high throughput sequencing platform. Taking advantage of the large sequence dataset now available, we analyzed features of these miRNAs, including miRNA sequence conservation through evolution, miRNA clusters, tissue-enriched expression, and sequence variations. Previously, using the 454 sequencing platform, we detected a Z chromosome encoded miRNA, miR-2954, in the male zebra finch brain. This miRNA has recently been shown to be expressed in the auditory forebrain of zebra finches, and its expression is regulated by song exposure. We have further explored the expression of miR-2954 and other Z chromosome encoded miRNAs in male and female tissues and have found that miR-2954 is predominantly expressed in all male tissues examined. Our target prediction analysis revealed that miR-2954, but not other Z chromosome encoded miRNAs, preferentially targets Z chromosome encoded genes.
General features of zebra finch miRNAs
miRNAs and their variants accounted for ~64% of all high quality reads. The remaining sequence reads were classified as rRNA/tRNA fragments, repeat-associated small RNAs, degradation products of mRNAs, and undefined small RNAs (Figure1B). The expression levels of individual miRNAs spanned a wide range, from a few copies to thousands of copies (Additional file2). For example, tgu-miR-1, which was highly expressed in both heart and muscle, was represented by 2,541,528 reads, accounting for 25% of the total miRNA reads (Figure1C), whereas the three novel zebra finch miRNAs were expressed at relatively low levels, represented by 304, 66, and 73 reads respectively (Figure1A). (Note, these numbers represent combined totals from four tissues and two sexes. For read counts in individual tissues and sexes, see Additional file2). All of the top 20 most abundantly expressed miRNAs were conserved in vertebrates, and they comprised 76% of the total miRNA sequence reads (Figure1C).
Potential avian specific miRNAs
Using a distance of 10 kb between any two miRNA genes as a cutoff, 87 miRNA genes were grouped into 36 genomic clusters. We named these miRNA gene clusters according to the name of the first member of each cluster followed by the number of miRNA genes within the cluster in parenthesis, e.g., cluster tgu-mir-24(3) contains tgu-mir-24, tgu-mir-27b, and tgu-mir-23b (Additional file4). Among these clusters, 3 appeared to be formed by tandem duplications of a single miRNA gene, e.g., the tgu-mir-2989(2) cluster on chromosome 8 contains 2 copies of the tgu-mir-2989 gene (Additional file4). A majority of clusters were conserved in vertebrates (having more than 2 miRNA genes in the same cluster in other species); one cluster (tgu-let-7a-2(3)) was also conserved in D. melanogaster, but none was conserved in C. elegans. One cluster (tgu-mir-2989(2)) appeared to be zebra finch specific.
We found 150 miRNA* sequences for 131 mature miRNAs (Additional file5). For miRNAs encoded by multiple genomic loci, a single mature sequence can have more than one star sequence, presumably originating from different precursors. For example, three different star sequences were detected for tgu-miR-7, which has three genomic loci. These three tgu-miR-7* sequences had different read counts, indicating that the three tgu-miR-7 genes were transcribed with different promoter activities and/or different efficacies of precursor processing (Additional file6). Most of the star sequences were detected at considerably lower levels compared to their respective dominant strands. Yet, for 16 miRNAs, both strands were detected at comparable levels (< 10 fold differences, Additional file7), suggesting that both strands may function as mature miRNAs. The relative read counts for the two strands of these miRNAs were similar across the four tissues examined (Additional file5), indicating that the mechanisms retaining both strands were tissue independent. Interestingly, for several miRNAs (e.g., tgu-miR-142, tgu-miR-214, and tgu-miR-455), both strands have been detected at similar levels in mice and chickens as well[12, 38], suggesting a functional conservation of these star sequences in vertebrates.
miRNAs generated by atypical biogenesis pathways
Mirtrons are a special group of miRNAs that are derived from short intronic sequences by splicing machinery rather than by Drosha cleavage[39–41]. Recently, Glazov and colleagues reported the identification of 12 mirtrons in chickens. We did not, however, find sequence homologs of any of the chicken mirtrons in our miRNA set, nor did we find any miRNAs mapping to either boundaries of a short intron. This discrepancy might be partly explained by the fact that the chicken mirtrons were detected in embryos and exhibited low copy numbers, whereas our miRNAs were from adult tissues. Alternatively, it may simply be a consequence of the incomplete annotation of intronic regions in the zebra finch genome.
miR-451 is a vertebrate miRNA whose maturation depends on Ago2 cleavage rather than the common Dicer pathway[42, 43]. We found that tgu-miR-451 was located on chromosome 19, about 100 nt downstream from miR-144, and the mature and precursor sequences of tgu-miR-451 were highly conserved with those in chickens, mice, and humans. Similar to its mammalian counterparts, the 5′-end of tgu-miR-451 was well defined, whereas its 3′-end was highly variable, extending into the loop region of the hairpin structure, presumably reflecting imprecise cleavage by Ago2 (Additional file8).
miRNA expression patterns in tissues
Conservation analysis of brain enriched miRNAs in three other species
Reads in brain
miRNAs within a genomic cluster tend to exhibit parallel relative expression patterns across tissues, probably reflecting their shared promoter and tissue specific cistronic transcriptional control. However, the expression abundance of different members of a cluster in a given tissue can be drastically different. For example, miR-133a and miR-1 of the 133a(2) cluster were both expressed in heart and muscle, but the expression of miR-1 was about 10 times higher than that of miR-133a in each of these tissues. This difference in expression levels between members was characteristic of many genomic clusters (Additional file9), suggesting that a precursor specific event during the miRNA maturation process might regulate the expression levels of individual miRNAs in a genomic cluster.
Untemplated nucleotide additions to miRNA 3' ends are observed for many miRNAs in worms, flies, and mammals[10, 12, 44, 45]. Similar to those observed in other species, nucleotides most frequently added to zebra finch miRNAs were U and A, with the U additions occurring more frequently than the A additions (66 miRNAs had U additions and 35 miRNAs had A additions, Additional file10). For some miRNAs, the extended isoforms outnumbered the canonical forms. For example, miR-456 had 7 fold more extended reads than non-extended reads, and miR-24 had 23,754 extended reads compared to 7,212 non-extended reads (Additional file10). We also observed that A addition had a greater tendency to occur on 5p arms, occurring 26 times on 5p arms compared to 9 on 3p arms, while U addition occurred slightly more often on 3p arms, occurring 36 times on 3p arms compared to 30 on 5p arms. Compared with previously published data, it appeared that patterns of untemplated extension of some miRNAs were conserved among multiple vertebrate species. For example, the A extensions of miR-99 and miR-101 and the U extensions of miR-15a, miR-24, miR-106, miR-124, and miR-425-5p were observed in zebra finch, human, and mouse[13, 46].
miRNAs having substitutions at the GGU motif
UGA GGU AG UAGGUUGUAUAGUU
UGA GGU AG UAGGUUGUGUGGUU
UGA GGU AG UAGGUUGUAUGGUU
AGA GGU AG UAGGUUGCAUAGUU
UGA GGU AG UAGAUUGAAUAGUU
UGA GGU AG UAGAUUGUAUAGUU
UGA GGU AG UAGUUUGUGCUGUU
UGGAGUGU GACAAUGGU GUUUG
UU GGU CCC CUUCAACCAGCUAU
ACCACAG G GU AGAACCACGGAC
UAGCAGCA CAUAAUGGU UUGU
UAGCAGCA CAUCAUGGU UUGU
AACAUUCA UUGCUGUCGGU GGGU
UAUGGCAC UGGU AGAAUUCACU
UAA GGU GC AUCUAGUGCAGAUA
UAA GGU GC AUCUAGUGCAGUU
UA GGU AGU UUCAUGUUGUUGGG
AGCUACAU UGUCUGCUGGGU UUC
AGCUACAU CUGGCUACUGGGU CUC
GACAGUCA GCAGUUGGU CUGG
AAUUGCAC GGU AUCCAUCUGU
CAGAUCAG AAGGU GAUUGUGGC
CAGGCUGG UUAGAUGGU UGUC
AUAGCUCU UGGAAUGGU UCUGC
GCGACCCA UACUUGGU UUCAG
Male-biased expression and targeting of miR-2954
Here we report a comprehensive annotation and analysis of 193 zebra finch miRNAs expressed in four different tissues of both sexes. While the majority of the 193 identified miRNAs are evolutionarily conserved, relatively large fractions of them are zebra finch-specific (10%) or avian-specific (15%). The list of zebra finch specific miRNAs may be even longer, as approximately 40% of the total sequence reads obtained from our small RNA libraries failed to map to the genome, probably due to gaps in the current genome assembly. Conversely, genomes of other species that we searched may have gaps as well, thus we cannot rule out the possibility that some avian and/or zebra finch specific miRNAs described here may have undiscovered homologs in other species. Several zebra finch specific miRNAs showed tissue specific expression. For example, miR-2963 was detected in the brain, miR-2997 in liver, and the novel miRNA 3 in heart (Additional file14). However, most of the zebra finch specific miRNAs were expressed at low levels, indicating that their expression is restricted to specific cell types or that they have not yet been widely incorporated into gene regulation networks. Further investigation will be needed to determine if these avian or zebra finch specific miRNAs play roles in zebra finch specific features.
Recently, Gunaratne et al. reported the annotation of 155 miRNAs expressed in the auditory forebrain region of zebra finches. Among these miRNAs, 140 are present in our set of miRNAs. Our dataset contains an additional 53 miRNAs, many of which show tissue specific expression in heart, liver, and muscle (Additional files2 and15). This is not unexpected as we sequenced four tissues, whereas only auditory forebrain was sequenced by Gunaratne et al. Although the source materials used in the two studies were different and the criteria for miRNA annotation were slightly different, the large overlap between the two datasets provides additional confidence with respect to the identification of these miRNAs. Combining the two sets of data brings the total number of zebra finch miRNAs to 208. Many of these miRNAs show enriched expression in the brain, including several (miR-25, miR-192, miR-124, miR-129, and miR-92) that are regulated in the auditory forebrain by song exposure, indicating that miRNAs may play important roles in song behavior and neural plasticity.
miRNA sequence heterogeneity
We did not observe significant A-to-I editing in mature zebra finch miRNAs except for miR-24. Notably, several mammalian miRNAs with well-characterized A-to-I editing sites (e.g., miR-376a, miR-376c, miR-379, miR-381, miR-411, miR-421, and miR-589,[12, 48, 49]) do not appear to have homologs in zebra finches, hinting that A-to-I editing might be more restricted to mammals. Instead, we observed internal nucleotide changes at the uridine of a GGU motif occurring at a relative high frequency. Our average base call sequencing error rate was <0.5% (calculated based on spiked-in synthetic internal controls), comparable to sequencing error rates observed by others. The rate of observed nucleotide changes at the GGU motif was as high as 14% (in miR-22). Thus it is unlikely that these changes were due to sequencing errors. We also examined each genomic locus for miRNAs known to have multiple genomic loci, and ruled out the possibility that they were due to genetic variations. We cannot rule out the possibility that these changes were due to single nucleotide polymorphisms (SNPs), as we currently know very little about SNPs in zebra finches. However, we would expect that the rate of nucleotide changes generated by SNPs would be much higher than we observed. To our knowledge, internal nucleotide changes at the uridine of a GGU motif has not been reported previously. Some of the GGU motifs are within seed sequences of mature miRNAs (e.g., members of the let-7 family). Nucleotide changes at these sites can change miRNA binding specificity, and subsequently, impact gene expression and related cellular processes. Further analysis will bring better understandings of biosynthesis mechanisms and potential biological functions of this type of internal nucleotide change.
Other types of sequence heterogeneity including untemplated 3' terminal extensions and length variations are far more prevalent than internal nucleotide changes. miRNA isoforms with 3' untemplated extensions account for ~15% of all miRNA reads. miRNA 3' untemplated A or U extensions have been widely reported by others in multiple animal species[12, 13, 52–56]. These events seem to be miRNA specific, as some miRNAs are more frequently extended than others. Interestingly, many of the zebra finch miRNAs showing high frequencies of 3' untemplated extensions are also frequently extended in other species (Additional file10,[11, 57, 58]), hinting that mechanisms governing the extension events and their potential functions are likely to be conserved. Although investigation of 3' untemplated extension is still at an early stage, 3' extension appears to be biologically regulated and to affect biological function. Emerging evidence suggests that 3' untemplated extensions provide a post-transcriptional mechanism to regulate miRNA stability and efficiency of target repression[57–59].
miR-2954 as a sex specific gene regulator in avian species
Despite extensive searching, we did not find sequence homologs of miR-2954 outside of the avian taxon. An extensive search among transcripts of crocodile and 11 bird species conducted by Gunaratne et al. found miR-2954 in several bird species but not in crocodile. Given that the genomes and transcriptomes searched in these studies are not complete, we cannot rule out the possibility that miR-2954 has as yet unidentified homologs in other species. However, its expression in chickens and zebra finches is now supported by three independent studies including ours[32, 35, 50]. The male-biased expression of miR-2954 in chicken embryos and in various zebra finch tissues suggests that it plays a role in sexually dimorphic animal development and function in avian species.
Animals adapt different dosage compensation mechanisms to balance the expression of sex chromosome genes between the two sexes and to balance the expression between sex chromosomal genes and autosomal genes[26, 60]. In avian species, females are heterogametic with one Z and one W chromosome, and males are homogametic with two Z chromosomes. Both the chicken and zebra finch lack a chromosome-wide dosage compensation mechanism, and many Z chromosome genes exhibit higher expression in males than in females[61–67]. While the male to female (M:F) expression ratios of most Z-linked miRNAs are close to 2, reflecting the copy number relationship, miR-2954 exhibits M:F expression ratios ranging from 6 to 20 (based on read counts) or 6 to 40 (based on qRT-PCR) in various tissues, which cannot be explained by the 2:1 difference in gene copy numbers. Clearly, additional male-biased factors contribute to the regulation of miR-2954 expression in various tissues.
In both chickens and zebra finches, dosage compensation can occur locally in a gene-specific manner[65–67]. In chickens, this is exemplified by dosage compensation mediated by the MHM (male hypermethylated) locus on nearby genes on the Z chromosome[26, 68, 69]. However, the zebra finch genome does not appear to contain the MHM locus, suggesting the existence of other sex specific dosage compensation mechanisms. The male-biased expression of miR-2954 and its preferential targeting of Z-chromosome genes may provide a novel dosage compensation mechanism at the post-transcriptional level. A miRNA-mediated gene-specific mechanism for sex chromosome gene regulation would offer flexibility in response to specific developmental and functional needs.
Both song behavior and the underlying neural circuit are highly sexually dimorphic in zebra finches. The song system nuclei HVC and the robust nucleus of the arcopallium (RA), which control motor patterns of song, are considerably larger in males than in females, and Area-X, a forebrain nucleus required for song learning, is a large nucleus in adult males but is almost invisible in adult females[27, 71]. The gene regulation network underlying this structural and functional sexual dimorphism is not clear. Among the putative targets of miR-2954, SCAMP1, a Z chromosome encoded synaptic vesicle associated protein gene, is known to be expressed at higher levels in HVC and RA in male zebra finches than in females. Recently, Gunaratne et al. reported that miR-2954 is regulated by hearing songs in the auditory forebrain of zebra finches, suggesting that in the context of song behavior this miRNA plays important roles in relaying physiological changes to changes in gene expression. Further analysis of miR-2954 expression in the song control circuits, and validation of its target genes will provide a better understanding of its roles in the sexually dimorphic structural and functional development of the zebra finch brain.
Our results provide a comprehensive miRNA expression atlas of brain, heart, liver, and muscle tissues of both male and female zebra finches. These data significantly enlarge the zebra finch miRNA repertoire, and will serve as a valuable resource for comparative and functional studies for the scientific community. In addition, we report a GGU motif as a potential site for miRNA internal substitution. We also describe male-biased expression of tgu-miR-2954, as well as its Z chromosome biased target relationship, which may point to a novel avian specific dosage compensation mechanism.
Library preparation and sequencing
Four tissues (heart, liver, muscle, and whole brain) from adult male and female zebra finches were collected, and total RNA was isolated using the Trizol method. The RNAs were used for library construction. Briefly, in a total reaction volume of 20 μl, 2 μg total RNA was ligated to 100 pmol adenylated 3′ adapter containing a unique pentamer barcode (App-(Barcode)TCGTATGCCGTCTTCTGCTTGT), 1 μg Rnl2(1–249)K227Q (plasmid for expression of recombinant ligase is available at http://www.addgene.org) in 50 mM Tris–HCl, pH 7.6; 10 mM MgCl2; 10 mM 2-mercaptoethanol; 0.1 mg/ml acetylated BSA (Sigma, St. Louis, MO), and 15% DMSO for 16 hours on ice. Following 3′ adapter ligation, 20 bar-coded samples were pooled and products were purified on a 15% denaturing polyacrylamide gel. Small RNAs, measuring 45–50 nt in length, were excised from the gel, eluted, and ligated to 100 pmol 5′ oligoribonucleotide adapter (GUUCAGAGUUCUACAGUCCGACGAUC) in a 20 μl reaction volume using 1 μg Rnl1 RNA Ligase in 50 mM Tris–HCl, pH 7.6; 10 mM MgCl2; 10 mM 2-mercaptoethanol; 0.2 mg/ml acetylated BSA; 0.2 mM ATP, and 15% DMSO for 1 h at 37°C. Ligated small RNAs were purified on a 12% polyacrylamide gel, reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA), and amplified by PCR using appropriate primers (forward primer: AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA; RT and reverse primer: CAAGCAGAAGACGGCATACGA). A total of eight libraries were barcoded and sequenced in one lane using Illumina GAII.
Sequence analysis and miRNA identification
After adapter trimming and removal of orphan reads, reads of 18–32 nt in length were kept for further analysis. For miRNA annotations, we compared sequences to known miRNAs (miRBase 17.0, 04/2011), and identical sequences were identified as homology miRNAs. Sequences were mapped to the zebra finch genome (Ensembl, taeGut3.2.4) permitting no mismatches. Sequences with more than 100 genomic loci were excluded from further analysis. Sequences homologous (with at most one mismatch) to known tRNA/rRNA/ncRNA sequences collected from the NCBI GenBank database were classified as tRNAs/rRNAs/ncRNAs. Small RNAs derived from repeat region/transposable elements were identified by screening the zebra finch genome using the RepeatMasker software. The remaining sequences were used for new miRNA candidate prediction. Flanking genomic sequences of various lengths (60, 80, 100, and 120 nt) of each small RNA mapping locus were extracted and subjected to analysis by the mFold program (http://mfold.rna.albany.edu/) to predict secondary structures. miRNA candidates were identified using the following criteria: (1) presence of hairpin-shaped precursor structures, (2) presence of >10 sequence reads, (3) presence of star sequences originated from the opposite stem of the hairpin structure, and (4) precise 5’ ends among all sequence variants. In addition, sequences homologous to known miRNAs in miRBase, without the star sequence were also accepted. Because the incompleteness of the current genome precluded unambiguous analysis, 16 miRNAs which did not meet criterion 1 but were homologous to known miRNAs were also accepted.
Sequence conservation analysis
We searched miRBase for known miRNA homologs in 9 species: chicken (galGal3), human (hg18), mouse (mm9), platypus (ornAna1), lizard (anoCar2), X. tropicalis (xenTro3), zebrafish (danRer7), drosophila (dm3), and C. elegans (ce10). We also compared zebra finch miRNA sequences to the genomes and ESTs (downloaded from the UCSC genome resources) of the same 9 species to search for homologs with at most 1 mismatch to the query sequence and presence of a hairpin shaped precursor structure. Qualified matches were identified as candidate miRNA homologs in the tested species. According to results based on these criteria, zebra finch miRNAs were classified into groups of zebra finch specific, avian specific, conserved in avian, human, and mouse, conserved in vertebrates, and conserved in all tested species.
We normalized the read counts of individual miRNA species to the total miRNA read number in each tissue to obtain an RPM measurement, ″R eads P er M illion reads″. RPM values for each miRNA in all tested tissues were compared. We defined a miRNA as ″highly enriched in one tissue″ if it had a minimum of 100 reads in all tissues, and its expression in one tissue accounted for ≥ 90% of all reads in all tissues combined.
miRNA sequence variant identification
All sequences passing the qualification filter were used for miRNA variant analysis. Length variants were selected among sequences matched perfectly to the zebra finch genome by comparing their lengths with the canonical miRNA sequences. For variants containing terminal modifications and internal substitutions, the NCBI Blastn program was used to compare all qualified sequence reads with identified miRNAs. Sequences matching multiple miRNAs were excluded to avoid ambiguity. The criteria used for accepting an internal nucleotide change variant included i) a requirement that the total number of sequence reads of a miRNA including its variants was > 100, ii) the number of reads with internal changes represented more than 5% of total reads, and iii) the mismatch occurred at positions at least 2 nucleotides from the 5’ and 3’ termini. A plot of the motif analysis (Figure4C) was generated with the WebLogo program (http://weblogo.berkeley.edu/).
Target gene prediction
For target gene prediction, the 3’ UTR sequences of all zebra finch and chicken genes were extracted from the Ensemble database (taeGut3.2.4 and WASHUC2) using the BioMart tool (http://www.ensembl.org/biomart/martview). Genes with 3’ UTR of less than 10 nt or without 3’ UTR were excluded. Since TargetScan (version 6.0,) does not support zebra finch sequences, we made minor modifications to adapt it for target analysis in the zebra finch. Genes containing target sequences within the 3’ UTRs that match perfectly to the 2–8 nt of a miRNA sequence were accepted as putative targets. Enrichment of miRNA targets on the Z chromosome was evaluated by Fisher′s exact test.
Northern blot and qRT-PCR
Total RNAs were extracted from relevant tissues of adult male and female zebra finches with Trizol reagent (Invitrogen). Total RNAs of chicken, human, and mouse were purchased from Ambion. For Northern blot analysis, 20 μg RNA from each tissue was separated on 20% denaturing gel, transferred to a nitrocellulose membrane, and hybridized to relevant miRNA probes labeled with 32P-dCTP at 65°C overnight. Hybridization signals were detected using a phosphorimager. For qRT-PCR, reverse transcription was performed using a custom-designed miR-2954 specific primer and the TaqMan microRNA reverse transcription kit (Applied Biosystems). qRT-PCR was performed in a 20 μL reaction volume containing 1.33 μL cDNA, 1 μL 20× custom designed miR-2954 Taqman probe, and the Taqman universal PCR master mix (Applied Biosystems) using conditions of 10 min at 95°C, followed by 40 cycles of 15 sec at 90°C, 1 min at 60°C. Relative miR-2954 expression was calculated by the ∆ ∆ Ct method as described. GAPDH was used as an internal control for normalizing miR-2954 expression, as no sex-based differential expression of GAPDH has been detected in zebra finches. All qRT-PCR reactions were run in triplicate.
The three novel zebra finch miRNAs have been assigned miRBase IDs as tgu-miR-7643 (novel 1), tgu-miR-7644 (novel 2), and tgu-miR-7645 (novel 3).
This study was supported by NIH grant MH081254 (XCL) and grants, 973 2011CBA01101 from the National Basic Research Program of China and KSCX2-EW-R-0103 from Chinese Academy of Sciences (XJW). MH was supported by the Charles Revson Jr. foundation. We thank Dr. C. Zhao and Genomic Core of the Rockefeller University for the sequencing work. We thank the Washington University Genome Center for sharing the zebra finch genome sequence prior to publication.
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