Identification and analysis of genetic variations in pri-miRNAs expressed specifically or at a high level in sheep skeletal muscle

Abstract

MicroRNAs (miRNAs) are key regulators in miRNA-mediated gene regulatory networks and play important roles in many biological processes, such as growth and development of mammals. In this study, we used microarrays to detect 261 miRNAs that are expressed in sheep skeletal muscle. We found 22 miRNAs that showed high levels of expression and equated to 89% of the total miRNA. Genetic variations in these 22 pri-miRNAs were further investigated using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing. A total of 49 genetic variations, which included 41 single nucleotide polymorphisms (SNPs) and 8 deletions/insertions, were identified in four sheep breeds. Three variations were further researched in a larger sample set, including five sheep breeds with different meat production performances. We found that the genotype and allele frequencies of the CCC deletion/insertion in pri-miR-133a were significantly related to the sheep meat production trait. Finally, cell assays and quantitative reverse transcription PCR (qRT-PCR) were employed to investigate the effect of pri-miRNA genetic variation on the miRNA biogenesis process. The results confirmed that genetic variations can influence miRNA biogenesis and increase or decrease the levels of mature miRNAs, in accordance with the energy and stability change of hair-pin secondary structures. Our findings will help to further the understanding of the functions of genetic variations in sheep pri-miRNAs in skeletal muscle growth and development.

Fig 1. Validation of the microarray results using qRT-PCR.

Five highly expressed miRNAs and three lowly expressed miRNAs were chosen according to the microarray dataset to detect their expression levels in sheep skeletal muscle. The graph was generated using MS Excel. Bar graphs show the mean ± SEM.

Fig 2. The characteristics of genetic variations in sheep pri-miRNA sequences.

(A) Frequency distribution of genetic variations in pri-miRNAs. (B) The location of genetic variations in pri-miRNAs. The 5′ nucleotide of pre-miRNA is +1, and the upstream or downstream locations of the variation are shown relative to the 5’ nucleotide. (C) Illustration of substitution types for SNPs that occurred in sheep pri-miRNAs. Arrows indicate the direction of the single nucleotide polymorphism.

Fig 3. The energy change distribution of secondary structures associated with genetic variations.

Fig 4. Illustration of pri-miR-133a (A), pri-let7a (B) and pri-miR-29c (C) secondary structure changes caused by genetic variations.

The box in (A) and circles in (B) and (C) mark the position of the genetic variations. Three typical pri-miRNAs secondary structures (parts of them) were found to display increased (ΔΔG = −1.6 kcal/mol), unchanged (ΔΔG = 0 kcal/mol) and decreased stability of hairpin structures (ΔΔG = 1.6 kcal/mol) as a result of the genetic variations.

Fig 5. PCR-SSCP and sequencing of pri-miR-133a (A), pri-miR-29a (B) and pri-miR-27b (C).

The boxes in the sequencing trace show the genetic variations. CCC (+/−) in (A) and TAATAATAC (+/−) in (B) appear mixed, with disordered peaks due to the deletion locus. GC in (C) appears as double peaks because the genetic variations occurred in one chromosome, but not the other.

Fig 6. Relative expression levels of pri-miR-133a (A), pri-miR-133b (B), pri-let7a (C), pri-miR-27b (D), pri-miR-29a (E) and pri-miR-128–2 (F) among the different genotypes of pri-miRNAs.

GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was chosen as the internal control gene. Seven genetic variations of six pri-miRNAs with different energy changes were chosen to investigate the effect of these energy changes on pri-miRNA expression in transfected HeLa cells. The expression level of each pri-miRNA showed no significant difference between allelic variants. Graphs were generated using MS Excel. Bar graphs show the mean ± SEM.

Fig 7. Relative expression levels of mature miR-133a (A), miR-133b (B), let7a (C), miR-27b (D), miR-29a (E) and miR-128–2 (F) among the different genotypes of pri-miRNAs.

miRNA biogenesis was affected in relation to the degree of energy change caused by the genetic variation. Graphs were generated using MS Excel. Bar graphs show the mean ± SEM.