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RNA N4-acetylcytidine (ac4C) modification is increasingly recognized as an important layer of gene regulation; however, the involvement of ac4C in pain regulation has not been studied. Here, we report that N-acetyltransferase 10 protein (NAT10; the only known ac4C “writer”) contributes to the induction and development of neuropathic pain in an ac4C-dependent manner. Peripheral nerve injury increases the levels of NAT10 expression and overall ac4C in injured dorsal root ganglia (DRGs). This upregulation is triggered by the activation of upstream transcription factor 1 (USF1), a transcription factor that binds to theNat10promoter. Knock-down or genetic deletion of NAT10 in the DRG abolishes the gain of ac4C sites inSyt9mRNA and the augmentation of SYT9 protein, resulting in a marked antinociceptive effect in nerve-injured male mice. Conversely, mimicking NAT10 upregulation in the absence of injury evokes the elevation ofSyt9ac4C and SYT9 protein and induces the genesis of neuropathic-pain-like behaviors. These findings demonstrate that USF1-governed NAT10 regulates neuropathic pain by targetingSyt9ac4C in peripheral nociceptive sensory neurons. Our findings establish NAT10 as a critical endogenous initiator of nociceptive behavior and a promising new target for treating neuropathic pain.SIGNIFICANCE STATEMENTThe cytidine N4-acetylcytidine (ac4C), a new epigenetic RNA modification, is crucial for the translation and stability of mRNA, but its role for chronic pain remains unclear. Here, we demonstrate that N-acetyltransferase 10 (NAT10) acts as ac4C N-acetyltransferase and plays an important role in the development and maintenance of neuropathic pain. NAT10 was upregulated via the activation of the transcription factor upstream transcription factor 1 (USF1) in the injured dorsal root ganglion (DRG) after peripheral nerve injury. Since pharmacological or genetic deleting NAT10 in the DRG attenuated the nerve injury-induced nociceptive hypersensitivities partially through suppressingSyt9mRNA ac4C and stabilizing SYT9 protein level, NAT10 may serve as an effective and novel therapeutic target for neuropathic pain.

Chronic inflammatory pain seriously affects patients’ quality of life because of a paucity of effective clinical treatments caused, at least in part, by lack of full understanding of the underlying mechanisms. miRNAs are known to be involved in inflammatory pain via silencing or degrading of target mRNA in the cytoplasm. The present study provides a novel mechanism by which miRNA-22 positively regulates metal-regulatory transcription factor 1 (Mtf1) in the nuclei of neurons in the dorsal horn of the spinal cord. We found that miRNA-22 was significantly increased in the dorsal horn of mice with either inflammatory pain induced by plantar injection of complete Freund’s adjuvant (CFA) or neuropathic pain induced by unilateral sciatic nerve chronic constrictive injury (CCI). Knocking down or blocking miRNA-22 alleviated CFA-induced mechanical allodynia and heat hyperalgesia, whereas overexpressing miRNA-22 produced pain-like behaviors. Mechanistically, the increased miRNA-22 binds directly to the Mtf1 promoter to recruit RNA polymerase II and elevate Mtf1 expression. The increased Mtf1 subsequently enhances spinal central sensitization, as evidenced by increased expression of p-ERK1/2, GFAP, and c-Fos in the dorsal horn. Our findings suggest that the miRNA-22–Mtf1 signaling axis in the dorsal horn plays a critical role in the induction and maintenance of inflammatory pain. This signaling pathway may be a promising therapeutic target in inflammatory pain.

Chronic inflammatory pain seriously affects patients' quality of life because of a paucity of effective clinical treatments caused, at least in part, by lack of full understanding of the underlying mechanisms. miRNAs are known to be involved in inflammatory pain via silencing or degrading of target mRNA in the cytoplasm. The present study provides a novel mechanism by which miRNA-22 positively regulates metal-regulatory transcription factor 1 (