Spatio-temporal regulation of gene expression is vital for normal growth and development in living organisms and for optimal response to environmental stimuli and endogenous cues. This regulation is achieved by multiple mechanisms operating at transcriptional, post-transcriptional and post-translational levels [1
]. Transcriptional regulation involving specific transcription factors interacting with their respective DNA cis
-elements and post-translational modifications involving protein modifications have been known for several decades. Only recently, the discovery of microRNAs (miRNAs; ~22-nt non-coding RNAs) has greatly expanded our knowledge of the cellular mechanisms that regulate gene expression at the post-transcriptional level in eukaryotes [3
]. Recent studies have suggested that miRNAs could serve as biomarkers for the identification of different types of cancers [5
] and/or as therapeutic targets or agents [6
Biochemical studies in animals support a compartmentalized, two-step maturation of miRNAs derived from their precursor transcripts originating from the miRNA genes located in the intergenic regions. Interestingly, many miRNAs in mammals are derived from the introns of the protein coding genes. The miRNAs are loaded into a silencing complex, called RNA-induced silencing complex (RISC) and can guide the complex to the target mRNAs. In most cases, animal miRNAs recognize their target transcripts by base pairing with the 7- to 8-nt complementary region usually located in the 3'UTRs on the target mRNAs and thus repressing their expression [8
], although mRNA cleavage might also occur [11
]. In contrast to the well-documented suppression of gene expression by miRNAs, miRNAs were recently suggested to also enhance target gene expression, and this "oscillation of activity between silencers to enhancers of gene expression" appears to depend on the state of the cell cycle [14
Since the initial discovery that lin-4 acts as a regulatory RNA in Caenorhabditis elegans
], interest in finding miRNAs and understanding their functions in diverse organisms has grown. Thus far, miRNAs have been shown to play critical roles in almost all biological processes examined, such as control of developmental timing, cell proliferation, cell fate specification, embryonic stem cell differentiation, limb development, adipogenesis, myogenesis, angiogenesis and hematopoiesis, neurogenesis, apoptosis, fat metabolism, insulin secretion, and even cancer [4
]. Many miRNA families are conserved among the vertebrate animals, and their functions may also have been well conserved. However, many of the new miRNAs recently discovered in human and chimpanzee are not conserved beyond mammals, and ~10% are taxon specific [17
]. As a result of extending this type of studies to more animal species, a number of lineage-specific miRNAs [18
] and species-specific miRNAs [18
] have been identified. These findings suggested that sequencing small RNA libraries from individual organisms is important to identify and catalog conserved and novel species-specific miRNAs. Computational approaches are effective in identifying conserved miRNAs in diverse plant or animal species [22
]. However, the process requires knowledge of the complete genome sequence, and species-specific miRNAs cannot be identified with confidence without this information. Direct small RNA sequencing is a straightforward and effective approach to characterize the miRNAs expressed from a genome of an organism [24
Pork, derived from pigs (Sus scrofa
), is one of the most widely eaten meats in the world [25
]. In recent years, the pig has been recognized as a potential model system for biomedical research, because pigs and humans have similarities in many aspects of their anatomy, physiology, biochemistry, pathology and pharmacology [25
]. Consequently, pigs can offer a system for understanding an array of health-related aspects of humans, such as obesity, diabetes, cancer, gastric ulcers, female reproductive health, cardiovascular disease, infectious diseases and organ transplantation [25
]. Additionally, pigs are closer evolutionarily to humans than mice [25
]. The economic and biomedical significance of pigs has led to the launch of the Swine Genome Sequencing Consortium (SGSC) [28
] to decipher the genome of the pig. The availability of the genome sequence and transcriptome analyses would significantly advance our ability to decipher various biological and biomedical secrets for better exploitation of the commercial traits of pigs as well as for the benefit of human health [26
]. Identification of miRNAs and their target genes can provide further insights into the post-transcriptional gene regulatory mechanisms influencing various biological and metabolic processes in pigs.
Bioinformatic approaches have been used previously to identify miRNAs in pig [25
] by exploiting the available genome sequence, 55 conserved miRNAs have been predicted (latest miRBase release 11.1 April, 2008). Recently, Kim et al. [29
] reported the identification of 17 new miRNAs belonging to conserved miRNA families in pig. However, this number is small compared to the several hundreds of conserved miRNAs known across the animal kingdom. In this study, we sequenced a small RNA library and experimentally validated 120 miRNAs. Only 24 matched the pig miRNAs listed in the miRBase (Table ). The remaining 96 miRNAs represent new miRNA homologs belonging to conserved miRNA families (Table ). Furthermore, we determined the temporal expression of 22 conserved and four pig-specific miRNAs in diverse tissues of pig.
Expression-based confirmation of previously predicted miRNAs in pig.
Newly identified miRNAs in pig that are homologous to known miRNAs from other animal species.