Discovery of miRNAs and Their Corresponding miRNA Genes in Atlantic Cod (Gadus morhua): Use of Stable miRNAs as Reference Genes Reveals Subgroups of miRNAs That Are Highly Expressed in Particular Organs

Abstract

BackgroundAtlantic cod (Gadus morhua) is among the economically most important species in the northern Atlantic Ocean and a model species for studying development of the immune system in vertebrates. MicroRNAs (miRNAs) are an abundant class of small RNA molecules that regulate fundamental biological processes at the post-transcriptional level. Detailed knowledge about a species miRNA repertoire is necessary to study how the miRNA transcriptome modulate gene expression. We have therefore discovered and characterized mature miRNAs and their corresponding miRNA genes in Atlantic cod. We have also performed a validation study to identify suitable reference genes for RT-qPCR analysis of miRNA expression in Atlantic cod. Finally, we utilized the newly characterized miRNA repertoire and the dedicated RT-qPCR method to reveal miRNAs that are highly expressed in certain organs.ResultsThe discovery analysis revealed 490 mature miRNAs (401 unique sequences) along with precursor sequences and genomic location of the miRNA genes. Twenty six of these were novel miRNA genes. Validation studies ranked gmo-miR-17-1—5p or the two-gene combination gmo-miR25-3p and gmo-miR210-5p as most suitable qPCR reference genes. Analysis by RT-qPCR revealed 45 miRNAs with significantly higher expression in tissues from one or a few organs. Comparisons to other vertebrates indicate that some of these miRNAs may regulate processes like growth, lipid metabolism, immune response to microbial infections and scar damage repair. Three teleost-specific and three novel Atlantic cod miRNAs were among the differentially expressed miRNAs.ConclusionsThe number of known mature miRNAs was considerably increased by our identification of miRNAs and miRNA genes in Atlantic cod. This will benefit further functional studies of miRNA expression using deep sequencing methods. The validation study showed that stable miRNAs are suitable reference genes for RT-qPCR analysis of miRNA expression. Applying RT-qPCR we have identified several miRNAs likely to have important regulatory functions in particular organs.

Discovery of microRNAs associated with the antiviral immune response of Atlantic cod macrophages

“…18 -24 nt) were mapped to a reference miRNAome (i.e. all of the known mature miRNAs in Atlantic cod) (Andreassen et al, 2016;Bizuayehu et al, 2015), using Novoalign (http://www.novocraft.com), and the mapped sequence reads were counted using a Custom-made script in Python. The reads mapped to the mature reference sequences with edit distance of one or less were considered true mature miRNAs.…”

“…While Atlantic cod miRNAs were previously discovered and characterised in early and later life stages (Andreassen et al, 2016;Bizuayehu et al, 2015), there was previously no information on cod miRNAs associated with antiviral immune responses.…”

miRNA analysis with Prost! reveals evolutionary conservation of organ-enriched expression and post-transcriptional modifications in three-spined stickleback and zebrafish

“…The hypothesis that miRNA functions are conserved predicts that at least some of the organ-enriched miRNAs in stickleback would also be enriched in the same organ in zebrafish. To test this prediction, we compared the list of organ-enriched miRNAs in stickleback and zebrafish and found that 44 miRNAs were brain-enriched in both species (Figure 3B, D, E), with many of them already known to be brain-associated miRNAs in several fish species 66–68 , for example miR9-5p, miR124-3p, and miR138-5p (Figure 4C-E), which are also highly expressed in brain and nervous organs in mammals 45,55,69–73 . These observations suggest a strong evolutionary conservation of function of brain-related, miRNAs among vertebrates.…”

“…Heart also displayed a significant number of evolutionarily-conserved, organ-enriched miRNAs (12 miRNAs) (Figure 3B, D, E). Heart-enriched miRNAs included mature products of the well-described vertebrate cardiac myomiR genes mir1, mir133 , and mir499 55,67,68,74–77 and erythromiRs mir144 and mir451 43 . The former group participate in muscle formation and function, and the latter may reflect the presence of red blood cells in the heart ventricle at the time of RNA extraction.…”

Profiling microRNA expression in Atlantic killifish (Fundulus heteroclitus) gill and responses to arsenic and hyperosmotic stress

“…A recent study reported 224 miRNAs in Atlantic salmon ( Salmo salar) blood, while the miRbase database lists 371 Atlantic salmon miRNAs, representing at least 11 tissues (Kozomara and Griffiths-Jones, 2014; Kure et al, 2013). More miRNAs have been identified in Atlantic cod ( Gadus morhua ), which has 401 miRNAs from at least nine tissues in miRBase (Andreassen et al, 2016; Kozomara and Griffiths-Jones, 2014). The number of miRNAs identified in these species is influenced by multiple factors, including the diversity of tissues examined, depth of sequencing, and the stringency of criteria used to define bona-fide miR As.…”