Identification and characterization of N6-methyladenosine circular RNAs in the spinal cord of morphine-tolerant rats

Abstract

Morphine tolerance (MT) is a tricky problem, the mechanism of it is currently unknown. Circular RNAs (circRNAs) serve significant functions in the biological processes (BPs) of the central nervous system. N6-methyladenosine (m6A), as a key post-transcriptional modification of RNA, can regulate the metabolism and functions of circRNAs. Here we explore the patterns of m6A-methylation of circRNAs in the spinal cord of morphine-tolerant rats. In brief, we constructed a morphine-tolerant rat model, performed m6A epitranscriptomic microarray using RNA samples collected from the spinal cords of morphine-tolerant rats and normal saline rats, and implemented the bioinformatics analysis. In the spinal cord of morphine-tolerant rats, 120 circRNAs with different m6A modifications were identified, 54 of which were hypermethylated and 66 of which were hypomethylated. Functional analysis of these m6A circRNAs found some important pathways involved in the pathogenesis of MT, such as the calcium signaling pathway. In the m6A circRNA-miRNA networks, several critical miRNAs that participated in the occurrence and development of MT were discovered to bind to these m6A circRNAs, such as miR-873a-5p, miR-103-1-5p, miR-107-5p. M6A modification of circRNAs may be involved in the pathogenesis of MT. These findings may lead to new insights into the epigenetic etiology and pathology of MT.

tRNA epitranscriptomic alterations associated with opioid-induced reward-seeking and long-term opioid withdrawal

“…RNAs that are single-stranded and covalently bound in a closed loop, which may be a molecular change contributing to the effects of morphine tolerance 10 . The m 6 A epitranscriptome was also implicated in alcohol use disorder (AUD), with a small cohort of human postmortem brains showing AUD-specific patterns of methylation at various mRNAs, long non-coding RNAs, and miRNAs 9 .…”

“…There is a wealth of evidence that chronic exposure to opioids induces dynamic and long-term changes to transcriptional machinery via epigenetic modifications to chromatin in the brain’s reward circuitry 4-7 , but very few studies have investigated the epitranscriptome in relation to addiction. To our knowledge, the only investigation into CNS epitranscriptomic changes in relation to opioids focused on m 6 A, showing that chronic morphine administration altered m 6 A profiles in spinal cord for many circular RNAs (circRNAs), non-coding RNAs that are single-stranded and covalently bound in a closed loop, which may be a molecular change contributing to the effects of morphine tolerance 10 . The m 6 A epitranscriptome was also implicated in alcohol use disorder (AUD), with a small cohort of human postmortem brains showing AUD-specific patterns of methylation at various mRNAs, long non-coding RNAs, and miRNAs 9 .…”

“…While some studies have investigated drug-induced changes in m 6 A, a modification most prominent on mRNA 9,10 , no studies have explored the role of m 5 C, a chemical modification most prominent on transfer RNAs (tRNAs), in addiction. tRNAs are integral for protein synthesis, a process altered by opioid exposure [11][12][13] , and m 5 C promotes tRNA stability in many mammalian tissues, including brain [14][15][16][17] .…”

“…These modifications are persistent, lending the ability to induce long-term dysregulation of neurobiological function which may serve as a largely unexplored mechanism mediating addiction vulnerability. While some studies have investigated drug-induced changes in m 6 A, a modification most prominent on mRNA 9,10 , no studies have explored the role of m 5 C, a chemical modification most prominent on transfer RNAs (tRNAs), in addiction. tRNAs are integral for protein synthesis, a process altered by opioid exposure 11–13 , and m 5 C promotes tRNA stability in many mammalian tissues, including brain 14–17 .…”

m6A modifications of circular RNAs in ischemia-induced retinal neovascularization

“…SuperScript TM III Reverse Transcriptase (Invitrogen) was used to synthesize the first-strand cDNA from IP RNA, and the system of QuantStudio TM 5 Real‑Time PCR (Applied Biosystems, Foster City, CA, USA) with 2X PCR master mix (Arraystar) was used to conduct RT-qPCR. The IP fraction in the input was calculated as MERIP/input (%) as described in Xing et al 23 . Data are presented as means ± SEM.…”