Assessing the effect of the CLPG mutation on the microRNA catalogue of skeletal muscle using high throughput sequencing

Abstract

The callipyge phenotype is a monogenic muscular hypertrophy that is only expressed in heterozygous sheep that receive the CLPG mutation from their sire. The lack of phenotypic expression in CLPG/CLPG animals is thought to result from the translational inhibition of paternally expressed DLK1 transcripts by maternally expressed miRNAs. To identify the miRNA responsible for this trans-effect we have used high-throughput sequencing to generate an exhaustive catalogue of miRNAs expressed in skeletal muscle of sheep of the four possible CLPG genotypes. We have identified 747 miRNA species of which 110 map to the DLK1-GTL2 or callipyge domain. We demonstrate that the latter are imprinted and preferentially expressed from the maternal allele. We show that the CLPG mutation affects their level of expression in cis (≈3.2-fold increase) as well as in trans (≈1.8-fold increase). In CLPG/CLPG animals miRNAs from the DLK1-GTL2 domain account for ≈20% of cellular miRNAs. We show that CLPG genotype affects the levels of A to I editing of at least five pri-miRNAs of the DLK1-GTL2 domain, but that levels of editing of mature miRNA are always minor. We present evidence that suggests that the miRNAs from the domain may target both the 3'UTR and ORF of DLK1 as a team thereby causing the trans-inhibition underlying polar overdominance. In this work we also highlight the quantitative limitations of high throughput sequencing for digital gene expression profiling as a result of biased and inconsistent amplification of specific miRNA species.