The development of next generation technologies is enabling the complete mapping and further understanding of microRNAs (miRNAs). Deep sequencing (NGS) provides complete coverage of the small transcriptome and new bioinformatics tools such as ACGT101-miR enable an exhaustive analysis of the sequencing data. The application of these new technologies together with the availability of a nearly complete pig genome has provided the basis for further defining the molecular and cellular function of these small regulatory molecules in the pig.
The domestic pig (Sus scrofa) is an important species from various standpoints. First, it is a major protein source in the human diet world-wide. Additionally, its anatomy, physiology, and genome size are very similar to the human species, and there has been increasing molecular genetic evidence showing the comparability of human and pig, making it a suitable model system for human biology. Pigs are now model animals for biomedical research of cardiovascular, immunological, cancer, diabetes, and a range of other diseases. Finally, the pig has become an important source of organs and tissue for transplantation into humans.
Recently, a world-wide collaboration of groups, from Houston, Texas to Sichuan, China, set out to establish a porcine miRNA atlas (microRNAome). The findings they report lay the groundwork for a greater understanding of the species through further mapping of tissue- and stage-specific miRNAs1.
Prior to this study, miRbase2, the primary public repository for miRNA sequence data, listed only 77 pig pre-miRs and 73 unique mature pig miRNAs; this out of a total of 10,883 database entries encompassing over 100 species. The number of pre-miRs for pig was significantly lower than for other species with similar size genomes (such as Human with 721 entries) suggesting the existence of far more pig miRNAs.
miRNA Analysis Bioinformatic Tool, ACGT101-miR
The researchers prepared 10 libraries of mixed tissue types for deep sequencing from 10 developmental stages representing the major morphological and physiological changes in pig growth from insemination through peak commercial value. The sequencing yielded approximately 10M reads per library sequenced. The ACGT101-miR bioinformatics package was employed to filter the reads for read quality, sequence pattern, copy number, read length, and other known RNA classes, and for mapping the quality reads to pig genome and mammalian miRNA sequence databases. Approximately 2.5M reads per library were mapped.
The ACGT101-miR bioinformatics tool provides:
- Mapping of all quality reads to custom databases
- Classification, alignment, length distribution & annotation of all mapped reads
- Prediction of possible novel miRs
- Detailed explanation of miRNA analysis results and their context
After mapping, the quality reads could be classified in one of four groups: reads that could be (1) mapped to known pig pre-miRNAs, (2) mapped to known other mammalian pre-miRNAs, (3) not mapped to any known pre-miRNAs, but mapped elsewhere in the pig genome or expressed sequence tags (ESTs) and predicted to form pre-miRNA hairpins, (4) not mapped to either pre-miRNAs or pig genome. Groups (1) and (2) were further divided into reads that also mapped to the pig genome or ESTs and reads that only mapped to the pre-miRNA.
Porcine miRNAome
Mining the significant amount of data generated in this study yielded:
771 - pig microRNAs
391 - conserved in mammals
380 - pig specific
When combined with the existing miRBase entries, the results of this study have expanded the coverage of the pig microRNAome to:
867 - pig pre-miRNAs
1004 - mature miRNAs
777 - unique mature miRNAs
The sequencing results of the ten libraries that span a pre- and post-natal time period not only greatly enriched our view of the pig microRNAome, but also provided information on time-dependent variations of the miRNAome as to sequence lengths, counts, composition, genomic location, and the relative expression of conserved versus pig-specific miRNAs. These results provide valuable sequence information about the porcine miRNAs which govern the functional activities of the miRNAome. Notably, a complete let-7 family of miRNAs were identified in this work, whereas the current miRBase v15 (April 2010) does not contain ssc-let-7b and ssc-let-7d. let-7 miRNAs are essential regulators of cell differentiation and have been linked to misregulation or misfunctioning of cell growth.
Expression Levels - A Global View
These new sequencing and bioinformatics technologies have enabled a much closer look at the pig microRNAome than was ever possible before. It is not surprising that those miRNAs that were found in high abundance had been mapped before, and indeed, the less abundant miRNAs were novel. According to the authors, the inability to previously detect these low abundance miRNAs was possibly due to their spatially, temporally, and physiologically transient expression or intermediate nature. Additionally, miRNA expression levels can provide clues as to their function. The top 10 miRNAs account for ~50% of all reads and the authors suggest that these highly expressed miRNAs may be involved in basic life functions/life cycle. The difference in expression levels of certain miRNAs pre and post birth is consistent with their well characterized functions. Two such differentially expressed miRNAs are the muscle specific miR-1a-3p which is important during embryonic development for muscle cell differentiation and the liver specific miR-122-5p which is essential to support a developing blood supply.
IsomiRs
The presence of isomer sequences increases the complexity of the functional roles of these regulatory molecules and documenting isomiRs is an important step towards understanding the target-specific roles of these varying sequences. The analysis tool, ACGT101-miR, employed in this study enabled the authors to precisely identify the copy numbers and relative abundances the isomiR sequences, information that is essential for detailed delineation of their functions. They found the presence of diverse sequence variants and that isomiRs may be present at different relative levels at different stages of life.
Distribution in Chromosomes
The location of the pre-miRNAs within the genome can also provide clues to their function, for example, closely located pre-miRNAs are candidates for co-regulation in their expression and gene targeting. X-linked pre-miRNAs are highly expressed in testis with targets related to cell cycle process during spermatogenesis. Most (623) of the pre-miRNAs detected in the study were mapped to coordinates in the known pig genome, and the clusters of pre-miRNA were more dispersed than is found in human, mouse or rat.
Sequence Conservation
Determination of sequence conservation revealed yet another clue to miRNA functions. The study found that conserved mammalian miRNAs were ubiquitously expressed at high levels in all stages of pig development. However, expression of pig specific miRNAs was much lower than the conserved sequences and appeared only at specific stages of development. The authors hypothesize that "the conserved miRNAs may be responsible for control of the basic cellular and developmental pathways common to most eukaryotes whereas the species-specific miRNAs may be involved in regulation of the lineage-specific pathways and functions".
The latest sequencing and bioinformatics technologies have opened a path for exploration and better understanding of pig molecular biology in growth and development, and for advanced pathway and function modeling in pig relevant to complex human diseases.
- Li M, Xia Y, Gu Y, Zhang K, Lang Q, Chen L, Guan J, Luo Z, Chen H, Li Y, Li Q, Li X, Jiang AA, Shuai S, Wang J, Zhu Q, Zhou X, Gao X, Li X. (2010) MicroRNAome of porcine pre- and postnatal development. PLoS One 5(7), e11541.
- Griffiths-Jones S, Grocock R, van Dongen S, Bateman A, Enright A. (2006) miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res 34(Database issue), D140-44.