Identification and characterization of 63 MicroRNAs in the Asian seabass Lates calcarifer

Abstract

Background: MicroRNAs (miRNAs) play an important role in the regulation of many fundamental biological processes. So far miRNAs have been only identified in a few fish species, although there are over 30,000 fish species living under different environmental conditions on the earth. Here, we described an approach to identify conserved miRNAs and characterized their expression patterns in different tissues for the first time in a food fish species Asian seabass (Lates calcarifer).

Methodology/principal findings: By combining a bioinformatics analysis with an approach of homolog-based PCR amplification and sequencing, 63 novel miRNAs belonging to 29 conserved miRNA families were identified. Of which, 59 miRNAs were conserved across 10-86 species (E value ≤ 10⁻⁴) and 4 miRNAs were conserved only in fish species. qRT-PCR analysis showed that miR-29, miR-103, miR-125 and several let-7 family members were strongly and ubiquitously expressed in all tissues tested. Interestingly, miR-1, miR-21, miR-183, miR-184 and miR-192 showed highly conserved tissue-specific expression patterns. Exposure of the Asian seabass to lipopolysaccharide (LPS) resulted in up-regulation of over 50% of the identified miRNAs in spleen suggesting the importance of the miRNAs in acute inflammatory immune responses.

Conclusions/significance: The approach used in this study is highly effective for identification of conserved miRNAs. The identification of 63 miRNAs and determination of the spatial expression patterns of these miRNAs are valuable resources for further studies on post-transcriptional gene regulation in Asian seabass and other fish species. Further identification of the target genes of these miRNAs would shed new light on their regulatory roles of microRNAs in fish.

Figure 1. The distribution of the seabass miRNA precursor candidates in various E value intervals as identified by the BLASTn program showing conservation with known pre-miRNAs.

Conservation of the 322 unique sequences that cloned from the seabass cDNA library with known pre-miRNAs was identified by the BLASTn program (E value). The most common conservation intervals were −2 (123 sequences), between e⁻² and e⁻³ (63 sequences) and followed by <e⁻⁸ (48 sequences).

Figure 2. Conservation of the miR-29 family in Pisces.

Predicted fold-back structures of mir-29 precursors from Lates calcarifer, Danio rerio, Fugu rubripes, Tetraodon nigroviridis and the folding energy (dG) for each fold-back structure are presented. The shadow in each precursor structure indicates the mature miR-29 sequence in the species.

Figure 3. Sequence logos showing the most conserved bases for miRNAs in one family.

The miRNA sequence logos for 15 lcal-miRNA families with multiple sequences (≥2) are presented. Each logo consists of stacks of symbols, one stack for each position in the sequence. The overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each nucleotide at that position. Some miRNAs from one family differ only by a few base pairs on either side and/or in the middle (e.g. let-7, mir-9, mir-21, mir-124 and mir-126); the mir-199 family is highly divergent, since the two miRNAs in that might be from the 5′ arm and from the 3′ arm of a pre-miRNA, respectively.

Figure 4. Conservation of the seabass mature miRNAs across species.

The 63 miRNAs validated by PCR and sequencing for the Asian seabass were searched against the miRBase database to identify the conservation across species. Of which, 59 miRNAs were conserved across 10–86 species and 4 miRNAs were found to be conserved less than 4 species according to our conservation criteria (E value≤10⁻⁴). Each small black rhombus represents an identified miRNA.

Figure 5. Expressions of the 57 conserved mature miRNAs in eight organs of the Asian seabass.

The expression of 63 miRNA was determined in 8 organs (gill, brain, eye, muscle, liver, intestine, heart and kidney) and in spleen sampled at 24 hour post challenge with LPS and control samples by real-time PCR. Six (lcal-miR-199a, lcal-miR-152, lcal-let-7a,b,c,d) of the sequencing validated miRNAs without positive results in all of the evaluated tissues were deleted from the data. Data were presented on a logarithmic scale. The relative expression of each gene shown in the figure was the average of triplicate real-time PCR reactions, normalized to lcal-miR-103 gene expression. Yellow shading indicated increased levels of expression, and gray shading represented decreased levels of expression relative to the center. Blue color denoted undetectable expression in tissues.