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

Abstract

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, Oprm1 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. Oprm1 circRNAs were sense-stranded circRNAs resistant to RNase R digestion. The relative abundance of Oprm1 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 circOprm1e2.3 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 Oprm1 circRNA sequences, suggesting a potential role in microRNA sequestration through sponging. In addition, we observed that other opioid receptor genes including δ, κ, 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. SIGNIFICANCE STATEMENT: The modulation of Oprm1 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 Oprm1 circRNAs in chronic opioid effects such as tolerance.

Fig. 1.

Mouse, rat, and human Oprm1/OPRM1 divergent RT-PCRs of circular RNAs. (A) A map of the mouse μ-opioid receptor gene (Oprm1) is shown with select exons in their relative locations (e.g., e1 designates exon 1). The e1 and exon 11 (e11) associated promoters are marked with bent arrows, and intron sizes are labeled in kilobases. The μ-opioid receptor (MOR-1) protein is encoded by the dominant linear mRNA containing e1-e2-e3-e4, shown above the genomic DNA (gDNA) with a polyA tail and locations of forward (F) and reverse (R) primers. The Oprm1 locus also generates multiple circRNA isoforms (shown below the gDNA map, with primer sites; circRNA sizes in nucleotides shown inside circles; red arrows point to backsplice junctions). (B) Divergent RT-PCR products from mouse [Mus musculus (Mm)] brain and spinal cord total RNA were Sanger sequenced and backsplice junctions confirmed (also see Supplemental Figs. 1 and 2). In brain, three variants were sequence confirmed, while only circOprm1.e2.e3 was confirmed in spinal cord. (C) Rat [Rattus norvegicus (Rn)] and (D) human [Homo sapiens (Hs)] divergent RT-PCRs were designed similarly to the mouse. Conventional RT-PCR with convergent primers (e2F-e3R) amplify linear mRNA as well as circRNAs, while divergent primers (e2F-e2R, e3F-e3R) only amplify circRNAs. In all cases, 5 μg total RNA was treated with reverse transcriptase [(RTase), arrow] with 250 ng random hexamers in 20 μl reactions (+), or RTase omitted (−). PCR was done with 1 ng/μl of cDNA input to 35 ampflication cycles. Ten microliters of PCR reaction were loaded against 1 μl of Invitrogen 1 kb Plus ladder; 500 bp (*) and 1 kb (#) are marked as size reference.

Fig. 2.

Mouse Oprm1 divergent RT-PCRs amplify sense-stranded circRNAs. (A) Primer maps with locations matching the MOR-1 mRNA and (B) dominant Oprm1 circRNAs are shown. Exons 1–5 are abbreviated e1–e5, and primer labels designate the target exons and orientation (F/R) with respect to transcription. The numbers inside the circles represent circRNA sizes (nt). (C) To confirm that divergent RT-PCR products are generated from circular RNAs, RNAs were pretreated with RNase R, an exonucleolytic enzyme that preferentially digests linear mRNA templates from the 3′ tails but not circular RNAs. Mouse brain total RNAs were digested with (+R) or without (−R) RNase R and samples were split to RT reactions with random hexamer (N6) primers, with (+) or without (−) reverse transcriptase (RTase) enzyme, and then these cDNAs were input to PCR using convergent or divergent primer pairs. (D) Convergent RT-PCR (primers e1F-e3R, lanes 1–4) amplified the linear MOR-1 mRNA, but RNase R predigestion attenuated this amplification. Divergent RT-PCR products (primers e2F-e2R, lanes 5–8) were stable against RNase R pretreatment. Divergent RT-PCR using primers from e1 to e2 (e2F-e1R primers, lanes 9–12) did not generate products. For labels above the gel image: top, middle, and bottom rows of labels represent RNase R treatment, inclusion/omission of RTase enzyme during RT with N6, and the PCR primer pairs used for each sample, respectively. For lane labels below the gel image, L represents the Invitrogen 1 kb plus ladder, and the 1 kb (#) and 500 bp (*) bands are marked. (E) Some circRNAs are known to derive from antisense transcripts overlapped with annotated genes; therefore, to determine whether the observed Oprm1 divergent RT-PCRs derive from sense or antisense circRNAs, RT reactions using strand-specific Oprm1 primers were performed as shown in the scheme. Mouse brain total RNA was incubated with either the Oprm1 e2 sense (e2F) or antisense (e2R) gene-specific primers, with (+) or without (−) RTase enzyme, and amplified with e2 divergent primers. (F) RT with antisense-stranded primer (e2R) can support divergent RT-PCR from sense-stranded circRNAs e2.e3, e2.e3.e5(119) and e2.e3.e4(302) (lanes 3 to 4), but sense-stranded primer (lanes 1 to 2) did not. Labels above the gel, from top to bottom, are for the RT primers, inclusion (+) or omission (−) of RTase enzyme, and PCR primers, while lane labels and size ladders are the same as for (D).

Fig. 3.

Mouse non-μ-opioid receptor divergent RT-PCRs. (A) A schematic of mouse μ (Oprm1), δ (Oprd1), κ (Oprk1), and nociceptin (Oprl1) receptor mRNAs for the dominant splice isoforms is shown, with exons labeled below (exon 1 is designated as e1). The mRNAs are organized by alignment of homologous exons, with transmembrane span coding regions colored black. Divergent RT-PCR primers for each target sequence are shown as arrows. Exons are not drawn to scale; narrow portions of exons represent untranslated regions. (B) Divergent RT-PCR was performed on mouse whole brain total RNA with PCR primer pairs directed outwardly from exons as labeled. For each gene, two primer pairs were used, targeting two separate exons. The Oprm1 locus generates several circRNAs, of which the e2.e3 circRNA appears dominant (*). Oprd1 e2 generates a single exon circRNA (dollar), and this template supported detection of a dimeric RT-PCR product consistent with processive reverse transcription of a single exon circRNA (#). Oprk1 also generates a single exon e3 circRNA (&). These divergent RT-PCRs were confirmed to derive from circRNAs by examination of backsplice sequences from direct Sanger sequencing, or after TOPO cloning, to confirm the existence of a backsplice junction and adherence to canonical GT-AG splice junctions. Five micrograms of total RNA were treated with reverse transcriptase using 250 ng random hexamers in 20 μl reactions, and 35 cycles of PCR amplification were done with 25 ng/μl input cDNA. Five microliters of PCR reaction were loaded against 1 μl of Invitrogen 1 kb Plus ladder; 500 and 1000 bp are labeled as size reference.

Fig. 4.

Anatomic survey of expression of mouse Oprm1 linear vs. circular RNAs. (A) The target Oprm1 RNAs are shown with exons and primers not drawn to scale. The linear Oprm1 isoforms are detected with primers spanning exon 1 (e1F1) to exon 3 (e3R2). The circOprm1.e2.e3 is detected with a backsplice junction spanning primer (e2e3juncR2) paired with an exon 3 forward primer (e3F1). The circOprm1.e2.e3.e4(302) is selectively amplified with two junction spanning primers. The backsplice junctions for circRNAs are shown with a red arrow. (B) ΔCt values for Oprm1 isoforms in opioid naive mouse whole brain and spinal cord are shown for linear, circOprm1.e2.e3, and circOprm1.e2.e3.e4(302). (C) Relative abundance values for circular isoforms normalized to the linear isoforms, using data shown in (B), are plotted as ΔΔCt values and analyzed by two-way ANOVA (significant variation was attributable to anatomic region, ***P < 0.001; RNA target, **P < 0.01; and interaction, **P < 0.01, see Materials and Methods). (D) Select brain regions were dissected from naive CD-1 male mice, and qPCRs were performed to evaluate relative expression of linear and e2.e3 circRNA levels (overall P < 0.05 by ordinary one-way ANOVA (F_6,14 = 2.953; *P = 0.045), followed by post-hoc multiple comparisons analysis with Tukey’s correction significant for cortex vs. brain stem (*P < 0.05).

Fig. 5.

circOprm1 RNAs accumulate in mouse whole brain with chronic morphine exposure. CD-1 male mice were subcutaneously implanted with a morphine [(M), 75 mg free base] or placebo (P) pellet every 3 days, for a total of three pellets/animal (n = 5 to 6 mice per group). Tissues were harvested on day 10 for qPCR analysis for Oprm1 RNAs to compare circRNA and linRNA levels in whole brain and spinal cord. Tissues were examined for circOprm1.e2.e3 (A), circOprm1.e2.e3.e4(302) (B), and linear mRNA (linOprm1.e1.e3) (C) levels. Data are shown as 2^−ΔCt. For each Oprm1 target RNA for each tissue, morphine vs. placebo comparisons were analyzed by t test, and circOprm1.e2.e3 circOprm1.e2.e3.e4(302) increases in whole brain were significant (*P < 0.05).

Fig. 6.

Predicted miRNA binding sites in mouse Oprm1 circRNAs. Potential miRNAs targeted by circOprm1.e2.e3 (A), circOprm1.e2.e3.e4(302) (B), and circOprm1.e2.e3.e5(119) (C) are shown. Shared miRNA binding sites are colored in gray, while sites unique for the circOprm1.e2.e3.e4(302) and circOprm1.e2.e3.e5(119) circRNAs are yellow. Circle sizes not to scale.

Fig. 7.

Predicted miRNA binding sites within human circOPRM1.e2.e3. A map of predicted miRNA binding sites within the human [Homo sapiens (Hs)] Hs.circOPRM1.e2.e3 is shown. Those miRNAs with experimental validation (in other contexts) are labeled in bold. Shown in orange are the predicted miRNA binding sites that are 100% conserved for the full heptamer miRNA seed sequence vs. the mouse homolog (Mm.circOPRM1.e2.e3). The red arrow depicts the location of the backsplice junction.

Fig. 8.

Post-transcriptional gene regulation by microRNAs and circular RNAs. Gene expression involves translation of mRNAs to proteins (arrow 1), but the process can be blocked when microRNAs bind to mRNAs through sequence complementarity (arrow 2). Depending on the microRNA sequences and the presence or absence of bulges in the binding due to the presence or absence of perfect matches in complementarity, mRNAs may become translationally silenced or targeted for degradation (arrow 2). However, circular RNAs also can bind and sequester microRNAs, lowering their ability to target mRNAs, a process termed sponging, which can block the inhibitory effects of microRNAs (arrow 3).