RNA sequencing reveals small RNAs differentially expressed between incipient Japanese threespine sticklebacks

Jun Kitano 0 1 Kohta Yoshida 1 Yutaka Suzuki 0 PRESTO, Japan Science and Technology Agency , Honcho Kawaguchi, Saitama 332-0012 , Japan 1 Ecological Genetics Laboratory, National Institute of Genetics , Yata 1111, Mishima, Shizuoka 411-8540 , Japan Background: 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. Results: 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. Conclusions: 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. - Background 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 [6]. 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 [9]. 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 2024 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 [12]. Another function of miRNA is epigenetic modification of genomic DNA: miRNAs interact with target DNAs to alter the chromatin state and suppress mRNA transcription [14]. 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 2432 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 (50U) [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 [26], 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 [35]. Additionally, cis-regulatory changes may include mutations at the target sites of small RNAs [36]; 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 [43]. 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 (...truncated)