Abstract
Circular RNAs (circRNAs) are ubiquitous endogenous RNA found in various organisms that can regulate gene expression in eukaryotes. However, little is known about potential roles for circRNAs in muscle development. We analyzed circRNA sequencing data of bovine skeletal muscle tissue and found differential expression of circTitin (circTTN) in fetal and adult bovine muscle tissue. We then further studied the role of circTTN in bovine muscle development. Overexpression and inhibition of circTTN together elicited its promoting roles in proliferation and differentiation of bovine primary myoblasts. Mechanistically, circTTN showed interaction with miR-432 by luciferase screening and RNA immunoprecipitation (RIP) assays. Additionally, miR-432 is a regulator of insulin-like growth factor 2 (IGF2), as indicated by luciferase activity, quantitative real-time PCR, and western blotting assays. Increased miR-432 expression inhibited the expression of IGF2, but this effect was remitted by circTTN. Conclusively, our results showed that circTTN promoted proliferation and differentiation of bovine primary myoblasts via competitively combining with miR-432 to activate the IGF2/phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway.
Highlights
Circular RNAs are widespread and diverse endogenous RNAs with covalently closed continuous loop structures, generated by back-splicing of a single pre-mRNA, making these RNAs more stable than linear RNA.[1,2] The first circRNA was identified in human cells in the early 1990s.3 circRNAs were discovered decades ago, they were originally considered byproducts of spliceosomemediated splicing errors and thought to lack any significant function.[4]
After treatment with RNase R, we found there was no significant decrease in circTTN expression, but the expression levels of TTN and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA were reduced (Figures 1D and 1E)
We investigated the stability of circTTN in bovine primary myoblasts
Summary
Circular RNAs (circRNAs) are widespread and diverse endogenous RNAs with covalently closed continuous loop structures, generated by back-splicing of a single pre-mRNA, making these RNAs more stable than linear RNA.[1,2] The first circRNA was identified in human cells in the early 1990s.3 circRNAs were discovered decades ago, they were originally considered byproducts of spliceosomemediated splicing errors and thought to lack any significant function.[4]. CircRNAs were discovered decades ago, they were originally considered byproducts of spliceosomemediated splicing errors and thought to lack any significant function.[4] recent high-throughput sequencing and novel computational approaches have identified a large number of circRNAs within the transcriptome, suggesting potential roles for these RNAs in development.[5,6,7,8,9] These circRNAs have extremely abundant microRNA (miRNA) binding sites and act as a competitive endogenous RNA (ceRNA) to regulate miRNA expression.[10,11] For example, two circRNAs, ciRS-7/CDR1as and Sry, were reported to contain multiple miRNA binding sites.[6,12] Besides, it has been proposed that some circRNAs may “sponge” other factors, such as RNA binding proteins (RBPs).[13] Recently, a novel subclass of circRNAs has been described as exon-intron circRNAs (EIciRNAs). These EIciRNAs interact with RNA polymerase II and U1 small nuclear ribonucleoproteins (snRNPs) and can act in cis to induce host-gene transcription in the nucleus.[14]
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