Alyref Deficiency Exacerbates Chronic Inflammatory Pain by Impairing Nuclear Export of Ccn3 MRNA

The 5-methylcytosine (m⁵C) RNA modification regulates multiple aspects of RNA metabolism; however, its contribution to pathological pain remains poorly understood. Here, we investigated the role of the m⁵C reader Alyref in a mouse (of either sex) model of complete Freund's adjuvant (CFA)-induced chronic inflammatory pain. We observed a marked and sustained upregulation of Alyref in nociceptive neurons of the dorsal root ganglion (DRG) following CFA administration. Conditional deletion of Alyref in Na_V1.8⁺ nociceptive neurons significantly exacerbated thermal and cold hypersensitivity, mechanical allodynia, and hyperalgesia. These behavioral abnormalities were accompanied by pronounced activation of microglia and astrocytes in the spinal dorsal horn, along with elevated expression of proinflammatory mediators, indicating enhanced neuroinflammation and central sensitization. Mechanistically, Alyref directly binds to m⁵C-modified Ccn3 mRNA and promotes its nuclear export, thereby maintaining cytoplasmic Ccn3 expression in DRG neurons. Loss of Alyref impaired Ccn3 mRNA export, reduced Ccn3 expression, and was associated with an increased level of MMP-9, a key mediator of neuroinflammatory pain signaling. Collectively, these findings identify Alyref as a protective regulator in chronic inflammatory pain by restraining neuroinflammation and limiting the emergence of neuropathic pain-like features. Our study highlights a previously unrecognized role of m⁵C-dependent RNA regulation in nociceptive sensitization and suggests Alyref as a potential therapeutic target for pathological pain.