PT  - JOURNAL ARTICLE
AU  - Takeshi Irie
AU  - Rebecca Shum
AU  - Ioanna Deni
AU  - Amanda Hunkele
AU  - Valerie Le Rouzic
AU  - Jin Xu
AU  - Roger Wilson
AU  - Gregory Fischer
AU  - Gavril W Pasternak
AU  - Ying-Xian Pan
TI  - Identification of abundant and evolutionarily conserved opioid receptor circular RNAs in the nervous system moduated by morphine
AID  - 10.1124/mol.118.113977
DP  - 2019 Jan 01
TA  - Molecular Pharmacology
PG  - mol.118.113977
4099  - http://molpharm.aspetjournals.org/content/early/2019/06/26/mol.118.113977.short
4100  - http://molpharm.aspetjournals.org/content/early/2019/06/26/mol.118.113977.full
AB  - 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 biological methods, Oprm1 circRNAs were detected in RNAs of rodent and human brains and spinal cords, as well as human neuroblastoma cells, suggesting evolutionary conservation. Sequencing confirmed bcksplicing using canonical splice sites. Oprm1 circRNAs were sense-stranded circRNAs resistant to RNase R digestion. The relative abundance of Oprm1 circRNA to linRNA determined by quantitative RT-qPCR 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 circOprm1.e2.3 and circOprm1.e2.e3.e4(302) levels by 1.5-1.6 fold relative to linRNA. Sequence analysis predicted numerous microRNA binding sites within Oprm1 circRNA sequences, suggesting a potential role in microRNA sequestration through sponging. In addition, we observed that other opioid receptor genes including delta, kappa, and nociceptin receptor genes produced similar circRNAs. In conclusion, all members of the opioid receptor gene family express circRNAs, with Oprm1 circRNA levels exceeding those of linear forms in some regions. The modulation of circRNA expression by morphine, coupled with their high abundance and existence of potential miRNA binding sites within circRNA sequences raise the question regarding the role of Oprm1 circRNAs in chronic opioid effects such as tolerance.SIGNIFICANCE STATEMENT The significances of this manuscript as it stands are four-fold: First, it is the first time that circRNAs of all opioid receptor genes were identified and characterized. Second, the Oprm1 circRNAs are abundant and conserved from rodent to human. Third, Oprm1 e2.e3 circRNA was differentially expressed among the brain regions. Finally, Oprm1 circRNAs were upregulated in a morphine tolerance mouse model, suggesting the potential roles of Oprm1 circRNAs in morphine tolerance. We believe that our findings in this manuscript, even without the functional evidence that we are currently working on, will provide novel insights into regulation and function of opioid receptor genes not only for the opioid research community, but also for the GPCR field at large. Additionally, our study also raises an important issue of differentiating linear mRNAs with circRNAs when analyzing gene expression in general.