Abstract
miRNAs have been widely investigated in terms of cell proliferation and differentiation. However, little is known about their effects on bird growth. Here we characterized the promoter of miR-206 in chicken and found that the preferable promoter was located in 1200 bp upstream of pri-miR-206. In this region, many key transcription factors, including MyoD, c-Myb, CEBPα/β, AP-4, RAP1, Brn2, GATA-1/2/3, E47, Sn, upstream stimulatory factor (USF) and CdxA, were predicted to bind and interact with miR-206 promoter. Overexpression of MyoD sharply increased miR-206 expression in both fibroblast and myoblast cells, and also the regulation in the myoblast cells was much stronger, indicating that miR-206 was regulated by MyoD combined with other muscle specific transcriptional factors. Aiming to further investigate the relationship between miR-206 mutation and transcriptional expression, total of 23 SNPs were identified in the two distinct bird lines by sequencing. Interestingly, the motif bound by MyoD was individually destroyed by G-to-C mutation located at 419 bp upstream of miR-206 precursor. Co-transfecting MyoD and miR-206 promoter in DF-1 cells, the luciferase activity of promoter containing homozygous GG types was significantly higher than CC ones (p < 0.05). Thus, this mutation caused low expression of miR-206. Consistently, eight variants including G-419C mutation exhibited a great effect on birthweight through maker-trait association analysis in F2 population (p < 0.05). Additionally, the regulation of miR-206 on embryo muscle mass mainly by increasing MyoG and muscle creatine kinase (MCK) expression (p < 0.05) with little change in MyoD, TMEM8C and myosin heavy chain (MHC). In conclusion, our findings provide a novel mutation destroying the promoter activity of miR-206 in birds and shed new light to understand the regulation mechanism of miR-206 on the embryonic muscle growth.
Highlights
MicroRNAs are defined as functional noncoding small RNAs, which mainly regulate target genes at transcriptional or post-transcriptional level by specially recognizing and binding to the untranslated regions or coding regions [1,2,3,4]
We found that the promoter activity of miR-206 mainly lies in about 1200 bp upstream regions of pre-miR-206 and sharply increased in myoblast cell compared to non-muscle fibroblast cell
Our results revealed the detailed characters of miR-206 promoter region and identified a causative mutation impacting on the expression level and growth performance in birds
Summary
MicroRNAs (miRNA) are defined as functional noncoding small RNAs, which mainly regulate target genes at transcriptional or post-transcriptional level by specially recognizing and binding to the untranslated regions or coding regions [1,2,3,4]. The classic mode of miRNA action is that miRNA binds to their targets and forms a stable RNA folding structure with much perfect complementarity in the seed sequences (2–8 nt at the 51 end) of miRNA, and induces mRNA degradation in vivo [5]. The miRNA merely inhibits the protein level with little mRNA change. Using next-generation sequencing to detect the miRNAs expression profile, 12 changed miRNAs were proved as biomarkers in the diagnosis of Alzheimer disease [9]. The patients with high-risk-score in their microRNA signatures had poor overall and disease-free survivals compared to the low-risk-score patients [10]. Until now little is known about the regulation mechanism of crucial miRNA genes on growth
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