MicroRNA identity and abundance in porcine skeletal muscles determined by deep sequencing

Abstract

MicroRNAs (miRNA) are short single-stranded RNA molecules that regulate gene expression post-transcriptionally by binding to complementary sequences in the 3' untranslated region (3' UTR) of target mRNAs. MiRNAs participate in the regulation of myogenesis, and identification of the complete set of miRNAs expressed in muscles is likely to significantly increase our understanding of muscle growth and development. To determine the identity and abundance of miRNA in porcine skeletal muscle, we applied a deep sequencing approach. This allowed us to identify the sequences and relative expression levels of 212 annotated miRNA genes, thereby providing a thorough account of the miRNA transcriptome in porcine muscle tissue. The expression levels displayed a very large range, as reflected by the number of sequence reads, which varied from single counts for rare miRNAs to several million reads for the most abundant miRNAs. Moreover, we identified numerous examples of mature miRNAs that were derived from opposite sides of the same predicted precursor stem-loop structures, and also observed length and sequence heterogeneity at the 5' and 3' ends. Furthermore, KEGG pathway analysis suggested that highly expressed miRNAs are involved in skeletal muscle development and regeneration, signal transduction, cell-cell and cell-extracellular matrix communication and neural development and function.

The Art of MicroRNA Research

“…The most abundant miRNA was miR-1, accounting for almost 90% of the reads, whereas the second most abundant miRNA was miR-206, with 5.7% of all reads. 48 As has previously been shown for human and rodent cell types, 49 -51 the sequences of the majority of the detected miRNAs indicated length and/or end-sequence variations. Based on predicted folding properties, the sequencing identified 35 potential novel miRNAs, although Northern blot analysis for 6 of these did not show the expected sizes for either a mature or precursor miRNA.…”

“…Based on predicted folding properties, the sequencing identified 35 potential novel miRNAs, although Northern blot analysis for 6 of these did not show the expected sizes for either a mature or precursor miRNA. 48 The sole report on cardiac deep sequencing was published by Rao et al using the Illumina platform to sequence small RNAs from male and female adult murine hearts. Although it was not disclosed how many different known and unknown miRNAs were detected, the authors showed that 40% of the cardiac reads were miR-1, with other abundant miRNAs being miR-29a, miR-26a, and let-7 family members.…”

Identification and abundance of miRNA in chicken hypothalamus tissue determined by Solexa sequencing

“…The data also highlighted pathways associated with Wnt signaling, which are conserved throughout evolution and regulate cell proliferation, morphology, motility, and fate during embryonic development. The ma�ority of the enriched KEGG pathways identified were also found to be overrepresented in a previous study of differentially expressed miRNA (Nielsen et al, 2010).…”

“…It can also detect miRNAs with very low copy numbers, which reflect either a low expression level in the tissue or a contribution from minor cell types such as blood or adipose or connective tissues in the biopsy specimen (Nielsen et al, 2010). Further experiments are in progress to identify novel miRNAs among the candidates reported herein.…”

“…The MAPK signaling pathway, which is a highly conserved energy sensor, regulates diverse cellular programs including growth, muscle, apoptotic responses, and apoptosis (Nielsen et al, 2010). The MAPK pathway is also related to other signaling pathways, such as 5'-adenosine monophosphate-activated protein kinase, Wnt, cell cycle, apoptosis signaling pathways, and others (Raman et al, 2007), which suggests that the genes involved in cell cycle progression and cell proliferation are targeted by miRNAs.…”