Identification of Abundant and Evolutionarily Conserved Opioid Receptor Circular RNAs in the Nervous System Modulated by Morphine.

Circular RNAs (circRNAs) are a distinct category of single-stranded, covalently closed RNAs formed by backsplicing. The functions of circRNAs are incompletely known and are under active investigation. Here, we report that in addition to traditional linear mRNAs (linRNA), mouse, rat, and human opioid receptor genes generate exonic circRNA isoforms. Using standard molecular biologic methods, circRNAs (circOprm1) were detected in RNAs of rodent and human brains and spinal cords, as well as human neuroblastoma cells, suggesting evolutionary conservation. Sequencing confirmed backsplicing using canonical splice sites. circRNAs were sense-stranded circRNAs resistant to RNase R digestion. The relative abundance of circRNA to linRNA determined by quantitative reverse transcription polymerase chain reaction varied among mouse brain regions, with circRNA isoforms predominating in rostral structures and less abundant in brain stem. Chronic morphine exposure in mice increased brain circe2.3 and circOprm1.e2.e3.e4(302) levels by 1.5- to 1.6-fold relative to linRNA. Sequence analysis predicted numerous microRNA binding sites within circRNA sequences, suggesting a potential role in microRNA sequestration through sponging. In addition, we observed that other opioid receptor genes including , , and nociceptin receptor genes produced similar circRNAs. In conclusion, all members of the opioid receptor gene family express circRNAs, with circRNA levels exceeding those of linear forms in some regions. SIGNIFICANCE STATEMENT: The modulation of circular RNA (circRNA) expression by morphine, coupled with the high abundance and existence of potential miRNA binding sites with circRNA sequences suggests the potential role of circRNAs in chronic opioid effects such as tolerance.