Identification of a novel circular RNA specific to the bovine placenta from the pregnancy-associated glycoprotein gene

<title>Abstract</title> <p>Circular RNAs (circRNAs) are covalently closed transcripts produced by back-splicing that regulate gene expression, including via microRNA (miRNA) sponge. However, trophoblast-specific circRNAs and their roles in the bovine placenta have not been well characterized. This study profiled circRNA expression in the bovine placenta during early gestation, validating abundant candidates and identifying circRNAs from trophoblast-specific genes. Sequencing of circRNAs from pooled placental tissues on day 65 of gestation revealed 13,958 circRNAs, 62% of which were derived from protein-coding regions, and were mostly 300–600 bases long. Highly expressed circRNAs were confirmed by divergent RT‒PCR, Sanger sequencing of back-splice junctions, and RNase R assays, which revealed the enrichment of circRNAs. These circRNAs and their host mRNAs exhibited largely ubiquitous tissue expression, with circRNA/host ratios ranging from ~ 5–6% to &gt; 20%. Among the circRNAs from trophoblast-specific genes, only bta_circ_2423 (from pregnancy-associated glycoprotein 4, PAG4) was confirmed to be circular and placenta-specific, showing a low circRNA/PAG4 mRNA ratio (0.85%) and peak abundance during mid-gestation. In silico analysis revealed that bta_circ_2423 binds to bta-miR-183, bta-miR-197, and bta-miR-3596, whose targets are enriched in membrane transport, metabolism, RNA regulation, and immune pathways. This study provides an early gestation bovine placental circRNA atlas and identifies a placenta-specific circRNA that potentially contributes to placental function via miRNA sponge activity.</p>