RNA sequencing reveals small RNAs differentially expressed between incipient Japanese threespine sticklebacks
© Kitano et al.; licensee BioMed Central Ltd. 2013
Received: 6 November 2012
Accepted: 20 March 2013
Published: 2 April 2013
Non-coding small RNAs, ranging from 20 to 30 nucleotides in length, mediate the regulation of gene expression and play important roles in many biological processes. One class of small RNAs, microRNAs (miRNAs), are highly conserved across taxa and mediate the regulation of the chromatin state and the post-transcriptional regulation of messenger RNA (mRNA). Another class of small RNAs is the Piwi-interacting RNAs, which play important roles in the silencing of transposons and other functional genes. Although the biological functions of the different small RNAs have been elucidated in several laboratory animals, little is known regarding naturally occurring variation in small RNA transcriptomes among closely related species.
We employed next-generation sequencing technology to compare the expression profiles of brain small RNAs between sympatric species of the Japanese threespine stickleback (Gasterosteus aculeatus). We identified several small RNAs that were differentially expressed between sympatric Pacific Ocean and Japan Sea sticklebacks. Potential targets of several small RNAs were identified as repetitive sequences. Female-biased miRNA expression from the old X chromosome was also observed, and it was attributed to the degeneration of the Y chromosome.
Our results suggest that expression patterns of small RNA can differ between incipient species and may be a potential mechanism underlying differential mRNA expression and transposon activity.
KeywordsStickleback Speciation Variation miRNA piRNA Ecology Variation
Recent progress in the development of genomic techniques, including next generation sequencers, has greatly facilitated transcriptome analysis of ecologically important animals to reveal variations in mRNA expression patterns among closely related species and ecotypes within species [1–3]. Divergence in mRNA expression patterns is known to contribute to phenotypic evolution [4, 5], although amino acid alterations in proteins are also important . While a great deal is known about variation in mRNA expression profiles, information regarding naturally occurring variation in the expression patterns of small RNAs is limited, except for a few cases in plants [7, 8] and cichlids .
Non-coding small RNAs, ranging from 20 to 30 nucleotides in length, mediate the regulation of gene expression [10–13]. The members of one class of small RNAs, microRNAs (miRNAs), are typically 20–24 nucleotides long and are highly conserved across diverse taxa [11, 12]. miRNA post-transcriptionally regulates messenger RNA (mRNA). A miRNA interacts with ten to hundreds of target mRNAs to induce degradation or suppress translation . Another function of miRNA is epigenetic modification of genomic DNA: miRNAs interact with target DNAs to alter the chromatin state and suppress mRNA transcription . miRNAs comprise more than 1% of animal genes [15, 16], suggesting that they play important roles in many biological processes. Recent functional studies in laboratory model animals such as mice, flies, and nematodes have demonstrated that miRNAs are important for regulating development, growth, pathogen resistance, and neural functions [11, 12, 17–19].
Another class of small RNAs is the Piwi-interacting RNAs (piRNAs), which are typically 24–32 nucleotides long and interact with Piwi proteins to suppress the expression of transposons and other functional genes [13, 20]. piRNAs often possess uridine at the 5’-end (5′U) [13, 20]. piRNAs are expressed from intergenic repetitive elements, active transposons, and piRNA clusters. Importantly, piRNAs may contribute to hybrid dysgenesis [21, 22]. For example, some Drosophila strains contain transposons as well as piRNAs that inhibit transposon activity, whereas other strains lack both transposons and inhibitory piRNAs. Because piRNAs are maternally transmitted, hybrid progeny resulting from a cross between a mother lacking both transposons and piRNAs and a father possessing both will inherit the transposons, but not the inhibitory piRNAs. This abnormal activity of transposons in the germ line is likely to result in sterility [21, 22]. Thus, maternally transmitted piRNAs can explain why hybrid abnormalities are observed in only one direction of the inter-strain crosses. piRNAs are expressed not only in the gonads, but also in the brain, and they may be involved in the regulation of neuronal functions [23–25]. Compared with miRNAs, piRNAs are less well conserved across taxa. Yet another class of small RNAs, endogenous small interfering RNAs (endo-siRNAs), are usually 21 nucleotides and have been found in some taxa, including nematodes , flies [27–29], and mammals [30, 31], but it has not been well characterized in other animals.
Evolutionary genetic studies examining small RNAs are important for several reasons. First, genome-wide allele-specific mRNA expression analyses have revealed that both cis- and trans-regulatory changes contribute to differential expression of mRNAs among closely related species [32–34]. Small RNAs can act as trans-regulatory factors, which contribute to differential mRNA expression . Additionally, cis-regulatory changes may include mutations at the target sites of small RNAs ; for example, SNPs and insertion-deletion polymorphisms were identified within miRNA-binding sites of 3’-untranslated regions [37, 38]. Variations in small RNA transcriptomes and sequences were found to be associated with phenotypic variation in humans and laboratory animals. For example, miRNA and miRNA target site polymorphisms and mutations have been found in humans and are associated with disease susceptibility [39–42]. Polymorphism in a miRNA target site is associated with variation of muscularity in pigs . Second, small RNAs regulate translation of mRNAs. Therefore, transcriptome studies of mRNA alone can overlook the divergence in the total outcome of gene expression among species. Third, piRNAs may contribute to hybrid abnormalities (see above), but generalities regarding the roles of piRNA in different types of hybrid abnormalities remain unclear.
In the present study, we compared brain small RNA transcriptomes between incipient species of the threespine stickleback (Gasterosteus aculeatus). The threespine stickleback is a good model for linking ecological and genetic studies of adaptive evolution and speciation [44–52]. The threespine stickleback has undergone tremendous diversification over the past few million years [44, 45, 49]. Evolutionary diversification within the stickleback species complex led to a speciation continuum, which ranges from populations with interspecific phenotypic polymorphism to strong divergence with near-complete reproductive isolation [44, 53]. Recent genetic studies have revealed that differences in the expression of genes involved in morphological development [54, 55], physiology [56, 57], and immune function  may underlie adaptive divergence among populations or species. Sex bias of the mRNA transcriptome has also been investigated, and genes located on sex chromosomes were found to be female-biased, likely owing to Y-chromosome degeneration and lack of dosage compensation . However, transcriptome analysis of small RNAs has not yet been conducted in any stickleback system.
This study focused on Japanese threespine stickleback species pairs, including a Pacific Ocean form and a Japan Sea form. These sticklebacks diverged during a period of geographical isolation between the Sea of Japan and the Pacific Ocean approximately 1.5–2 million years ago [60, 61]. Currently, they are sympatric in eastern Hokkaido, but they are reproductively isolated with a very low level of hybridization [60–62]. In the Japan Sea form, a chromosomal fusion occurred between linkage group (LG) 9 and the ancestral Y chromosome (LG 19), resulting in the evolution of the X1X2Y multiple sex chromosome system . In contrast, the Pacific Ocean form has a simple XY sex chromosome system . Previously, we found that the Pacific Ocean and Japan Sea forms diverge in male courtship behaviors and female mate choice behaviors, contributing to behavioral isolation between these two forms [60, 62, 63]. Furthermore, we found that divergence in the intensity of courtship behaviour, which is important for mate choice, mapped to a neo-sex chromosome (LG 9).
To better understand the genetic mechanisms affecting behavioral differences between this Japanese stickleback species pair, it is essential to understand divergence in small RNA transcriptomes of the brain. Both miRNAs and piRNAs play important roles in a diverse array of neuronal functions such as neuronal differentiation, neural stem cell renewal, neuronal outgrowth, and dendritic spine morphogenesis [23, 24, 64]. Furthermore, variation in miRNA expression patterns in the brain may contribute to behavioral differences among laboratory mouse strains . Additionally, in the Japanese stickleback system, courtship dysfunction may exist in hybrids because a substantial number of hybrids did not perform any courtship behavior in a previous experiment (Supplementary data in ). Therefore, it is necessary to examine whether small RNAs, especially piRNAs, which may be regulating transposon activity in the brain, affect hybrid behavior. Here, we characterized the divergence in small RNA transcriptomes in the brain between the species pairs of Japanese threespine stickleback.
Results and discussion
miRNA differentially expressed between species
Median reads per million (RPM) ± S.D.
Pacific Ocean males
Pacific Ocean females
Japan Sea males
Japan Sea females
ANOVA of miRNA
Species X Sex
miRNA differentially expressed between males and females
Median reads per million (RPM) ± S.D.
Pacific Ocean males
Pacific Ocean females
Japan Sea males
Japan Sea females
Because miRNAs can target many mRNAs [75, 76], divergence in miRNA expression patterns may have substantial effects on the expression patterns of many mRNAs. Further experimental studies examining the roles of small RNAs in fish will be necessary to understand the functional effects of the miRNA transcriptome variation. This would be possible by developing either transgenic fish specifically overexpressing small RNAs or small RNA-deficient knockout fish [77–81].
Small RNAs homologous to repetitive sequences
Small RNAs with nucleotide lengths larger than 25 nt
Hit in the piRNABank
Characterization of small RNAs with high homology to repetitive sequences
Blast hits (E < 10-4)
Potential target repeat
Median reads per million (RPM) ± S.D of the longest isoform
Pacific Ocean male
Pacific Ocean female
Japan Sea male
Japan Sea female
LG1(2), LG7(2), LG8(3), LG10(1), LG11(7), LG15(1), LG16(1), LG17(1), scaffolds(86)
LG3(1), LG7(1), LG11(2), LG12(1), LG17(5), LG18(1), LG19(1), LG20(1)
LG3(54), LG5(1), LG9(1), LG11(2), LG12(1), LG13(1), LG17(1), LG19(1), LG20(32), scaffolds(30)
LG5(1), LG9(1), LG11(2), LG12(1), LG13(1), LG17(1)
LG8(1), LG9(1), LG11(1), LG12(1), LG13(1), LG16(1), scaffolds(5)
LG7(6), LG18(10), scaffolds(44)
LG1(1), LG4(1), LG7(1), LG12(1), scaffold(1)
ERV1-type endogenous retrovirus
LG3(1), LG7(1), LG11(2), LG12(1), LG17(5), LG18(1), LG19(1), LG20(1)
LG3(1), LG7(1), LG11(2), LG12(1), LG17(5), LG18(1), LG19(1), LG20(1)
ERV1-type endogenous retrovirus
The remaining six and three isoforms overlapped with repetitive sequences homologous to tRNA and rRNA, respectively. Previous studies also identified a number of tRNA-derived small RNAs in humans [83, 84], Giardia lamblia, and zebrafish . These tRNA-derived small RNAs may contribute to gene regulation , although little is yet known about their functions. Interestingly, some transposons are derived from tRNAs [87, 88], so there appears to be an interesting link between tRNA and small RNAs.
Finally, we found that the iso-smRNA9, whose potential targets are predicted to be non-LTR transposons, was more highly expressed in the Japan Sea sticklebacks than in the Pacific Ocean sticklebacks (ANOVA, F1, 13 = 14.5, P = 0.002). Although expression levels of some other piRNAs may differ between different species and sexes, the differences were not significant after Bonferroni correction (Additional file 5: Table S5). None of the non-annotated small RNAs showed sex differences in the expression levels (Additional file 5: Table S5).
Thus, we identified small RNAs homologous to repetitive sequences such as RNA and DNA transposons. Hybrids between species often exhibit courtship dysfunction [89–94]. Abnormal transposon activity in hybrids may cause hybrid courtship dysfunction, but this has not been tested in any organism. Intra- and inter-population variation in the presence and absence of non-LTR retrotransposons has been found in sticklebacks . In addition, our analysis involving whole genome sequence comparisons also revealed that DNA transposon insertion sites diverge between the Pacific Ocean and the Japan Sea sticklebacks (Kitano, unpublished data). Therefore, further studies examining variation in transposon activity between the different species and transposon activity in the hybrids will lead to a better understanding of speciation mechanisms.
Our study demonstrates that closely related species can show divergence in expression patterns of small RNAs, including miRNAs and piRNAs. Some of the sex differences in miRNA expression levels might result from Y-chromosome degeneration. Therefore, variation in small RNA transcriptomes should be examined as a potential mechanism underlying phenotypic divergence between incipient species.
Small RNA sequencing
Sympatric Pacific Ocean and Japan Sea sticklebacks were collected using minnow traps from the Bekanbeushi River System on Hokkaido Island, Japan in June 2007 [60, 62]. Fish were brought to a laboratory to examine the brain transcriptome of courting male and spawning female fish, and mating experiments were conducted in June and July 2007, as described previously [60, 63, 96]. Once the male fish had constructed a nest, a conspecific gravid female fish was placed in the same tank. Immediately after the female fish had inspected the nest, both the male and the female fish were removed from the tank prior to spawning. After immersing the fish in a lethal dose of tricaine methanesulfonate, the brains of each fish were dissected and stored separately at −70°C.
For RNA sequencing, we used four Pacific Ocean male, four Pacific Ocean female, four Japan Sea male, and four Japan Sea female fish (N = 16 in total). Total RNA was isolated using TRIzol Reagent (Life Technologies, Grand Island, NY, USA), and the quality of RNA was evaluated using the BioAnalyzer (Agilent, Santa Clara, CA, USA). RNA Integrity Number (RIN) ranged from 9.3 to 10 with the median of 10. Libraries were constructed using the TruSeq Small RNA Sample Preparation Kit (Illumina, San Diego, CA, USA). Small RNAs with 20–30 nucleotides were isolated according to the manufacturer’s instructions. Sequencing was performed using the Genome Analyzer IIx (Illumina, San Diego, CA, USA) at the University of Tokyo. We used 16 lanes of the Genome Analyzer IIx (one fish per one lane).
Sequence analyses were conducted using the CLC Genomics Workbench Software (CLC bio, Katrinebjerg, Denmark). First, we discarded reads with a low quality score (quality score on the Phred scale of less than 0.05), very short length (less than 14 bp), or three or more ambiguous nucleotides (Additional file 1: Table S1). Next, identical reads were clustered together to group different types of small RNAs. Next, the sequences were mapped against the Ensembl stickleback miRNA database (http://asia.ensembl.org/info/data/ftp/index.html) with two nucleotides mismatches allowed.
Principal component analysis (PCA) of reads per million (RPM) was conducted on a Pearson correlation matrix. To identify differentially expressed miRNAs between various species and sexes, statistical analyses were conducted on the square-root transformed RPM of each miRNA. Using the statistical package R , analysis of variance (ANOVA) was conducted to examine whether the species, sex, and their interactions significantly influenced RPM. Because the patterns of sex differences varied between species (see Figure 3), we included the interaction term for the analysis. The Bonferroni correction was used for multiple comparison correction. Shapiro-Wilk and Bartlett’s tests were used to test for normal distribution of the data and homogeneity of variances, respectively. None of the miRNAs violated the assumptions of the normal distribution or homogeneity of variances after Bonferroni correction. Only miRNAs with a mean RPM higher than 1000 in at least one of the four groups (Pacific Ocean male, Pacific Ocean female, Japan Sea male, or Japan Sea female) were used for PCA and ANOVA.
Small RNAs with no match to any sequences in the stickleback miRNA database were mapped against sequences in the Ensembl stickleback non-coding RNA database (http://asia.ensembl.org/info/data/ftp/index.html), which includes transfer RNA (tRNA), ribosomal RNA (rRNA), small cytoplasmic RNA, small nuclear RNA, small nucleolar RNA, microRNA precursors, long intergenic non-coding RNAs, and other miscellaneous RNA. The parameters used were the same as above. Small RNAs with no match to any sequences in the stickleback non-coding RNA database may contain piRNAs; thus, we further analyzed these small RNAs to identify stickleback piRNAs. We first examined non-annotated small RNAs with nucleotide lengths >25 because this fraction is more likely to contain piRNAs. Among these longer small RNAs, we examined small RNAs with a mean RPM higher than 50 in at least one of the four groups (Pacific Ocean male, Pacific Ocean female, Japan Sea male, or Japan Sea female). Although some small RNAs showed differences in length, identical sequences were observed (Additional file 3: Table S3); hence, identical reads of different sizes were considered to be the same isoform.
To examine whether these small RNAs contain homologous sequences against any repetitive sequences, the longest isoforms were blasted against the stickleback genome (BROADS 1.56) using the default parameters (match = 1; mismatch = −3; gap existence = 5; gap extension = 2) of the CLC Genomics Workbench software. Next, flanking sequences (3,000 bp of upstream and 3,000 bp of downstream) were downloaded using Perl script . We then examined whether these regions contained any repetitive sequences using the CENSOR software  on the Genetic Information Research Institute website (http://www.girinst.org/). When hits of repetitive sequences were identified in the region, we investigated whether the small RNA sequences overlapped with repetitive sequences. We also blasted these sequences against the piRNABank database .
To identify differentially expressed piRNAs between different species and sexes, statistical analysis was conducted on the square-root transformed RPM. Square-root transformed RPM values were subjected to ANOVA, followed by Bonferroni correction. Shapiro-Wilk and Bartlett’s tests were used to test for normal distribution of the data and homogeneity of variances, respectively. Two small RNAs, iso_smRNA3 and iso_smRNA14, did not meet the assumptions of homogeneity of variance (Bartlett’s test, K-squared = 15.2, P = 0.0017) and normal distribution (Shapiro-Wilk test, W = 0.779, P = 0.0014), respectively. For these small RNAs, we also conducted a Mann–Whitney U-test to confirm that these two small RNAs did not exhibit any differences between sexes and species. Because the interaction between the sexes and different species showed no significant effect for any of these small RNAs, the interaction term was excluded.
Additional files: Tables S1-S5. All of the short read sequences are deposited in the Sequence Read Archive (DRA) of the DNA Data Bank of Japan (DDBJ): accession number DRA000919.
reads per million
principal component analysis
analysis of variance
This research is supported by the JST PRESTO program and Grant-in-Aid for Scientific Research on Innovative Areas ‘Gene Correlative System’ (23113007 and 23113001) and ‘Genome Science’ (221S0002) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Animal use protocols were approved by the Institutional Animal Care and Use Committee of the Fred Hutchinson Cancer Research Center (1575).
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