Abstract
Circular RNAs (circRNAs) have been identified from various tissues and species, but their regulatory functions during developmental processes are not well understood. We examined circRNA expression profiles of two developmental stages of bovine skeletal muscle (embryonic and adult musculus longissimus) to provide first insights into their potential involvement in bovine myogenesis. We identified 12 981 circRNAs and annotated them to the Bos taurus reference genome, including 530 circular intronic RNAs (ciRNAs). One parental gene could generate multiple circRNA isoforms, with only one or two isoforms being expressed at higher expression levels. Also, several host genes produced different isoforms when comparing development stages. Most circRNA candidates contained two to seven exons, and genomic distances to back-splicing sites were usually less than 50 kb. The length of upstream or downstream flanking introns was usually less than 105 nt (mean≈11 000 nt). Several circRNAs differed in abundance between developmental stages, and real-time quantitative PCR (qPCR) analysis largely confirmed differential expression of the 17 circRNAs included in this analysis. The second part of our study characterized the role of circLMO7—one of the most down-regulated circRNAs when comparing adult to embryonic muscle tissue—in bovine muscle development. Overexpression of circLMO7 inhibited the differentiation of primary bovine myoblasts, and it appears to function as a competing endogenous RNA for miR-378a-3p, whose involvement in bovine muscle development has been characterized beforehand. Congruent with our interpretation, circLMO7 increased the number of myoblasts in the S-phase of the cell cycle and decreased the proportion of cells in the G0/G1 phase. Moreover, it promoted the proliferation of myoblasts and protected them from apoptosis. Our study provides novel insights into the regulatory mechanisms underlying skeletal muscle development and identifies a number of circRNAs whose regulatory potential will need to be explored in the future.
Highlights
Prevents the application of several analytical methods that are widely used in RNA biology
We identified a considerable number of RNAs in embryonic and adult bovine muscle tissue when considering total RNA libraries (Table 1)
In the libraries that were depleted of ribosomal RNA, we identified 12 981 circRNAs candidates, 1287 of which contained at least one unique back-spliced read (Supplementary Table S2), including 530 circular intronic RNAs
Summary
Prevents the application of several analytical methods that are widely used in RNA biology. CiRS-7 and Sry inhibit miRNAs in murine tissue by sponging miR-7 and miR-138, suggesting that circRNAs may play important roles in post-transcriptional gene regulation.[7,12] circRNAs may sponge RNA-binding proteins. As another example, circMbl is derived from the muscleblind locus (MBL/MBNL1)— a splicing factor in Drosophila melanogaster—and contains MBL binding sites. Our present study was intended to serve as a starting point for future studies examining the role played by circRNAs in bovine muscle development. In order to reduce false positives arising from the RNA library treatment and the applied method to detect circRNAs, the RNAs of several samples were pooled and afterwards prepared as rRNA−+ RNase R+ libraries to investigate circRNA contents
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