Functional divergence of the rapidly evolving miR-513 subfamily in primates
© Sun et al.; licensee BioMed Central Ltd. 2013
Received: 13 August 2013
Accepted: 14 November 2013
Published: 19 November 2013
The miR-513 subfamily belongs to an X-linked primate-specific miR506-514 cluster. Across primate species, there have been several duplication events and different species each possess a variety of miR-513 copies, indicating it underwent rapid evolution. Evidence suggests that this subfamily is preferentially expressed in the testis, but otherwise, to date, the evolutionary history and functional significance of this miRNA subfamily has remained largely unexplored.
We analyzed the evolutionary pattern of gene duplications and their functional consequence for the miR-513 subfamily in primates. Sequence comparisons showed that the duplicated copies of miR-513 were derived from transposable element (MER91C). Moreover, duplication events of the miR-513 subfamily seem to have occurred independently in Platyrrhini (New World monkeys) and Catarrhini (Old World monkeys, apes and humans) after they diverged. Different copies of the miR-513 subfamily (miR-513a/b/c) have different seed sequences, due to after-duplication sequence divergences, which eventually led to functional divergences. The results of functional assays indicated that miR-513b could inhibit the expression of its target gene, the down-regulator of transcription 1 (DR1) at both the mRNA and protein levels. In the developing testis of rhesus macaques, we observed a temporal coupling of expression levels between miR-513b and DR1, suggesting that miR-513b could affect male sexual maturation by negatively regulating the development-stage related functioning of DR1.
The miR-513 subfamily underwent multiple independent gene duplications among five different lineages of primates. The after-duplication sequence divergences among the different copies of miR-513 led to functional divergence of these copies in primates.
MicroRNAs (miRNAs) are small (~22nt) endogenous non-coding RNAs that function as post-transcriptional gene regulators of a variety of biological processes [1–3]. Most miRNAs discovered at the early stage are highly conserved among species, indicating strong functional constraints on miRNA evolution. However, an increasing number of rapidly evolving miRNAs have been discovered and experimentally verified . In an evolutionary study, Bentwich et al. reported two miRNA clusters in primates with more copies than those present in rodents, suggesting miRNA gene expansion during primate evolution. One of the two clusters, the miR-506-514 cluster, is located on the X chromosome and is preferentially expressed in testis . Zhang et al. reported that this cluster has undergone rapid evolution in primates, with frequent gene duplications and nucleotide substitutions. In rhesus macaques, for example, expression analysis revealed a strong correlation between miRNA expression changes and male sexual maturation . Unfortunately, to date, the functional significance of these duplicated miRNAs has not been experimentally established.
In the miR-506-514 cluster, the miR-513 subfamily has the greatest diversity in terms of both copy number and sequence variations . There are also between-copy sequence substitutions in the mature miRNAs, especially in the seed region, which are critical for gene targeting. These observations collectively suggest that members of the miR-513 subfamily may play different roles in gene regulation.
In the most general sense, studying non-conserved miRNA may help to clarify how miRNAs originate and evolve, and likewise contribute to functional divergence among species. In the present study, we investigated the molecular mechanisms underlying the frequently observed gene duplications and sequence divergences in the miR-513 subfamily, and evaluated the impact of these sequence substitutions on target gene regulation and functional diversification.
MiR-513 is a rapidly evolving miRNA subfamily in primates
Functional divergence among gene copies of the miR-513 subfamily
Expression of GNG13, DR1 and BTG3 in testis of rhesus macaques
MiR-513b down-regulates the expression of DR1 at mRNA and protein levels
MiR-513 is an X-linked miRNA subfamily with multiple copies that has undergone rapid evolution in primates. Sequence comparisons revealed that the duplicated copies of miR-513 were derived from transposable elements (MER91C), different from other members within the miR-506-514 cluster. Zhang et al. showed that the duplications of another subfamily (miR-514) in this cluster occurred independently in humans and chimpanzees . By contrast, the initial duplication event of the miR-513 subfamily seems to have occurred earlier in the common ancestor of Catarrhini. Together, these results indicated that the miR-513 subfamily has a unique evolutionary history, quite different from other members in the same miRNA cluster.
Different copies of the miR-513 subfamily (miR-513a/b/c) with diverged seed sequences seem to have led to functional divergences. Our results showed that miR-513a, miR-513b and miR-513c are capable of specifically targeting their predicted sites in the 3′UTRs of GNG13, DR1 and BTG3 respectively. GNG13 is a guanine nucleotide binding protein subunit, expressed in taste, retinal, and neuronal tissues, and earlier studies reported GNG13 playing a key role in taste transduction . In contrast to one or several miRNA binding sites in the 3′ UTRs of target genes, there are 13 target sites of miR-513a in the 3′ UTR of GNG13, because the 3′ UTR of GNG13 contains 13 repeat elements matching the recognition site of miR-513a, resulting in a strong miR-513a-dependent inhibition of luciferase expression. Additionally, Gong et al. reported that miR-513a could regulate B7-H1’s translation and is involved in the IFN-gamma signal pathway in cholangiocyte . These findings suggest that the functional role of miR-513a is likely diverged from miR-513b/c and may not be related to male reproduction—an implication of neo-functionalization.
DR1 (down-regulator of transcription 1) is a TBP (TATA box-binding protein) associated phosphoprotein that regulates both basal and activated levels of transcription . BTG3 is a member of the BTG3/Tob family that has antiproliferative properties . Previous reports offered no substantive proof as to whether DR1 or BTG3 is involved in testicular development. However, over the course of our study we observed significant up-regulation of both DR1 and BTG3 in the testis after sexual maturation of male rhesus macaques, and a negative correlation between the expression of DR1/BTG3 and miR-513, suggesting miR-513′s role on testicular development via its regulation of DR1 and BTG3. Moreover, we showed that miR-513b could inhibit the expression of DR1 in vitro at both the mRNA and protein levels. TBP (a DR1 associated protein) has been reported to play an important role on spermatogenesis . Taken together, these findings prompt us to propose that miR-513b affects male sexual maturation by negatively regulating the development-stage-related functioning of DR1. Although there is no solid functional evidence, miR-513c emerged only in apes and humans, but shown sequence divergence in different lineages, implying that it may contribute to lineage-specific functions.
In summary, we demonstrated that the miR-513 subfamily underwent multiple independent gene duplications among different lineages of primates. More importantly, the after-duplication sequence divergences among the copies of miR-513 have led to functional divergence of these copies in primates.
Sequence analysis and target gene prediction
MiR-513 precursor and mature sequences were retrieved from the miRBase database (http://www.mirbase.org) and remapped to reference genomes of four primate species in UCSC using BLAT with default setting, including gorilla (gorGor3), orangutan (ponAbe2), gibbon (nomLeu3), and mouse lemur (micMur1). The genomic locations and sequences of the miR513s were compared to transposable elements (TEs) annotated with the RepeatMasker program (http://www.repeatmasker.org/). The criteria were ≥50% sequence identity and ≥80% location overlap. For phylogenetic analysis, precursor sequences were aligned by ClustalW (Gap opening 15; Gap extension 6.66; Transition Weight: 0.5) . Phylogenetic trees were reconstructed using the neighbor-joining method and maximum likelihood in MEGA5 and evaluated with 1,000 bootstrap replications . AnGST was also used to construct the gene tree by merging an ensemble of trees from different bootstrapping into a single “chimeric” tree (http://almlab.mit.edu/angst/). Tree ensemble used for AnGST was generated by the non-parametric bootstrapping step of PhyML (1,000 bootstrap replications) (http://www.atgc-montpellier.fr/phyml/). In order to infer gene duplication and loss events, gene tree and species tree reconciliation was performed by AnGST and Notung (http://www.cs.cmu.edu/~durand/Notung/). The species tree was based on the NCBI taxonomy tree (http://www.ncbi.nlm.nih.gov/taxonomy). To identify potential targets of miR-513 sequences, we used TargetScanS , and eight genes with top rank values were selected for functional testing.
All the constructs for this study were derived from psiCHECK™-2 vector (Promega, C8021). Human 3′UTR sequences were amplified from genomic DNA of human white blood cells using PCR. The oligonucleotide primers used for the generation of constructs are shown in Additional file 1: Table S2. PCR products were sub-cloned into psiCHECK™-2 vector. All constructs were then verified by sequencing. All synthesized miR-513a/b/c mimics (miR10002877, miR1000578 and miR10005789) and the mimic negative control (miR01201) were purchased from RiboBio (China).
Cell culture, transfection and tissue preparation
HeLa and HEK293T cells were grown in high glucose DMEM containing 10% FBS (HyClone, Logan, USA). They were transiently transfected with DNA constructs and miRNA mimics at 40% confluence in 6 or 24 well plates using Lipofectamine 2000 (Invitrogen). Cells were lysed 24–72 hours after transfection for luciferase assay, real time PCR, or western blotting. Three infant (~2 years old) and three adult (~8 years old) rhesus macaque testis samples were used in this study.
After transiently transfected with DNA constructs and miRNA mimics, HeLa or HEK293T cells are lysed by Reporter Lysis Buffer for 10–20 min. The luciferase activity in cell extracts was determined by Dual-luciferase Reporter Assay System (Promega, E1910) according to the manufacturer’s protocols. The relative light units were measured using a luminometer.
Real time quantitative PCR
Total RNA was isolated from tissues or transfected cells. These RNAs were reverse-transcribed with oligo-dT (20) primer and amplified by real time primers. Real time quantitative PCR (qPCR) reactions (15 μl total volume containing 0.5 μl 10 μM primer, 7.5 μl SYBR Green dye (Bio-Rad, CA, USA), and 2 μl of cDNA) were carried out with a DNA Engine Opticon Continuous Fluorescence Detection System (MJ Research Inc, Waltham, MA) for 40 cycles. The Ct values for each gene amplification were normalized by subtracting the Ct value calculated for ATCB. Normalized gene expression values were taken as the relative quantity of BTG3 or DR1 gene-specific messenger RNA (mRNA). The oligonucleotide primers used in the qPCR amplifications are shown in Additional file 1: Table S3.
Protein from transfected cells was homogenized in RIPA buffer (Beyotime, P0013) containing a cocktail of protease inhibitor (Sigma Chemical, MO, USA). Extracted protein (15–20 μg) was separated by SDS-polyacrylamide gel electrophoresis and transferred to a membrane by electrophoretic transfer. The membrane was incubated withβ-actin monoclonal antibody (Abcam, ab6276, 1:5000), anti-BTG3 polyclonal antibody (Abcam, ab112938, 1:500) and anti-DR1 polyclonal antibody (Abcam, ab88597,1:500). Then the membrane was incubated with HRP-conjugated secondary antibodies. Immunoreactivity was detected with an enhanced chemiluminescence system (Pierce, IL, USA) with colored markers (Fermentas) as molecular size standards.
Availability of supporting data
Phylogenetic data used in this study were submitted to TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S14738?x-access-code=9db8bc2ae28ac495564c94409d1023e8&format=html).
Many thanks to Hui Zhang for her technical help in this study. This work was supported by grants from the National 973 project of China (2012CBA01304) and the National Natural Science Foundation of China (31130051).
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