Evolution of the myosin heavy chain gene MYH14 and its intronic microRNA miR-499: muscle-specific miR-499 expression persists in the absence of the ancestral host gene

Abstract

Background: A novel sarcomeric myosin heavy chain gene, MYH14, was identified following the completion of the human genome project. MYH14 contains an intronic microRNA, miR-499, which is expressed in a slow/cardiac muscle specific manner along with its host gene; it plays a key role in muscle fiber-type specification in mammals. Interestingly, teleost fish genomes contain multiple MYH14 and miR-499 paralogs. However, the evolutionary history of MYH14 and miR-499 has not been studied in detail. In the present study, we identified MYH14/miR-499 loci on various teleost fish genomes and examined their evolutionary history by sequence and expression analyses.

Results: Synteny and phylogenetic analyses depict the evolutionary history of MYH14/miR-499 loci where teleost specific duplication and several subsequent rounds of species-specific gene loss events took place. Interestingly, miR-499 was not located in the MYH14 introns of certain teleost fish. An MYH14 paralog, lacking miR-499, exhibited an accelerated rate of evolution compared with those containing miR-499, suggesting a putative functional relationship between MYH14 and miR-499. In medaka, Oryzias latipes, miR-499 is present where MYH14 is completely absent in the genome. Furthermore, by using in situ hybridization and small RNA sequencing, miR-499 was expressed in the notochord at the medaka embryonic stage and slow/cardiac muscle at the larval and adult stages. Comparing the flanking sequences of MYH14/miR-499 loci between torafugu Takifugu rubripes, zebrafish Danio rerio, and medaka revealed some highly conserved regions, suggesting that cis-regulatory elements have been functionally conserved in medaka miR-499 despite the loss of its host gene.

Conclusions: This study reveals the evolutionary history of the MYH14/miRNA-499 locus in teleost fish, indicating divergent distribution and expression of MYH14 and miR-499 genes in different teleost fish lineages. We also found that medaka miR-499 was even expressed in the absence of its host gene. To our knowledge, this is the first report that shows the conversion of intronic into non-intronic miRNA during the evolution of a teleost fish lineage.

Figure 1

Genomic organization of MYH14 and miR-499 in various vertebrates. Orthologous genes are connected by solid and dotted lines. Genes displayed above the midline are in forward strands (+ orientation, from left to right), whereas those displayed below are in reverse strands (− orientation, from right to left). MYH14 and miR-499 paralogs found in one species are distinguished by numbers (see Table 1). Abbreviations used: Chr, chromosome; TRPC4AP, transient receptor potential cation channel, subfamily C, member 4 associated protein; EDEM2, ER degradation enhancer, mannosidase alpha-like 2; SLA2, Src-like-adaptor 2; NDRG3, N-myc downstream regulated family member 3; PHF20, PHD finger protein 20; SULF2, sulfatase 2.

Figure 2

MYH14 and miR-499phylogenetic analysis.MYH14(A) and miR-499 (B) neighbor-joining (NJ) trees. Bootstrap values from 1000 replicate analysis are given at the nodes as percentage values. Black circles indicate duplication of the MYH14/miR-499 locus.

Figure 3

miR-499 expression in medaka. Whole mount of a medaka embryo at 5 days post fertilization (dpf) (A) and a hatching larva at 10 dpf (B). miR-499 transcripts were detected in the notochord of the embryo and in cardiac and trunk skeletal muscles in the hatching larva. C) Ventral view of miR-499 expression in the heart of a 10-dpf larva. D) Transverse section of cardiac muscle at the position indicated in panel B. E) Transverse section from trunk skeletal muscle at the position indicated in panel B. Arrows indicate miR-499 expression in superficial slow muscle fibers. Transverse sections of adult cardiac (F) and trunk skeletal muscles (G). H) Higher magnification of the square indicated in panel G. miR-499 was expressed in cardiac but not in trunk muscle at the adult stage. I) miR-499 expression confirmed by next-generation sequencing. Vertical axis indicates miR-499 read numbers in each tissue. Scale bars: A-C, 500 μm; D-H, 200 μm.

Figure 4

Medaka miR-499 characteristics. (A) Comparison of the flanking and related sequences of torafugu MYH14-1 (MYH_M5) with zebrafish MYH14-1 and medaka miR-499. Highly conserved (>75%) regions between the two sequences are indicated by red-shaded peaks. Several highly conserved regions were identified at the MYH14/miR-499 5′-flanking and intron, as shown in blue boxes. (B) Putative secondary structures of mirtron (Caenorhabditis elegans miR-62) and miR-499.

Figure 5

Putative evolutionary history of MYH14 and miR-499 in the fish lineage. The common ancestor of amniotes and fish had a single miR-499 containing MYH14. Neoteleostei-specific whole genome duplication formed two sets of MYH14/miR-499 pairs. In the zebrafish lineage, additional tandem duplication resulted in three MYH14/miR-499 pairs. In torafugu, green spotted puffer, and tilapia, redundancy in miR-499 caused the deletion of one of the two miR-499 paralogs. In the stickleback and Atlantic cod lineage, an additional gene loss occurred in one of the two MYH14 paralogs and loss of the remaining MYH14 gene resulted in its complete elimination from the medaka genome.