Abstract
The domestic pig (Sus scrofa) is an important economic animal for meat production and as a suitable model organism for comparative genomics and biomedical studies. In an effort to gain further identification of miRNAs in the pig, we have applied the Illumina Solexa sequencing technology to carry out an in-depth analysis of the miRNA transcriptome in a pool of equal amounts of RNA from 16 different porcine tissues. From this data set, we identified 437 conserved and 86 candidate novel miRNA/miRNA* in the pig, corresponding to 329 miRNA genes. Compared with all the reported porcine miRNAs, the result showed that 112 conserved and 61 candidate novel porcine miRNA were first reported in this study. Further analysis revealed extensive sequence variations (isomiRs) of porcine miRNAs, including terminal isomiRs at both the 5′ and 3′ ends and nucleotide variants. Read counts of individual porcine miRNA spanned from a few reads to approximately 405541 reads, confirming the different level of expression of porcine miRNAs. Subsequently, the tissue expression patterns of 8 miRNAs were characterized by Northern blotting. The results showed that miR-145, miR-423-5p, miR-320, miR-26a, and miR-191 are ubiquitously expressed in diverse tissues, while miR-92, miR-200a, and miR-375 were selectively enriched and expressed in special tissues. Meanwhile, the expression of 8 novel porcine-specific miRNAs was validated by stem-loop RT-PCR, and one of these was detected by Northern blotting. Using the porcine miRNA array designed according to our Solexa results, 123 miRNAs were detected expression in porcine liver tissues. A total of 58 miRNAs showed differential expression between the Tongcheng (a Chinese indigenous fatty breed) and Large White pig breeds (a lean type pig). Taken together, our results add new information to existing data on porcine miRNAs and should be useful for investigating the biological functions of miRNAs in pig and other species.
Highlights
MicroRNAs belong to a group of single-stranded noncoding RNAs that are 21–23 nucleotides in length [1]
A typical example is miR-196, which can direct the cleavage of HOXB8 mRNA in mouse embryos [8]. miRNA biogenesis occurs through a multistep process that involves the activities of two Ribonuclease IIIs called Drosha and Dicer [9,10]
Overview of the Solexa sequencing data To increase the coverage of porcine miRNAs by Solexa sequencing, a small RNA library was constructed from the pooled porcine RNA samples collected from 16 tissues of young and adult pigs
Summary
MicroRNAs (miRNAs) belong to a group of single-stranded noncoding RNAs that are 21–23 nucleotides (nt) in length [1]. The first characterized miRNA was lin-4 in 1993, but miRNAs were not recognized as a distinct class of biological regulators with conserved functions until the early 2000s [2,3,4] These molecules typically interact with target mRNAs through base pairing, predominantly between bases 2–8 (the ‘‘seed’’ region) at the 59 end of the miRNA and complementary sites in the target 39 untranslated regions (39 UTR, ‘‘seed matches’’) [5]. These interactions result in translational repression and/or deadenylation of target mRNA [6,7]. A single miRNA can have one to several hundred target mRNAs, and it is estimated that 30% of all protein-coding genes in humans are regulated by miRNAs [5]
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