Nonsense-mediated decay constrains nociceptive responses through repression of the integrated stress response

RNA stability is meticulously controlled. Here, we sought to determine if an essential post-transcriptional regulatory mechanism plays a role in pain. Nonsense-mediated decay (NMD ) safeguards against translation of mRNAs that harbor premature termination codons and controls the stability of roughly 10% of typical protein-coding mRNAs. It hinges on the activity of the conserved kinase SMG1. Both SMG1 and its target UPF1 are expressed in sensory neurons throughout the dorsal root ganglion (DRG). SMG1 protein is present in DRG neurons and their fibers in the sciatic nerve. Using high-throughput sequencing, we examined changes in mRNA abundance following inhibition of SMG1. Among the characteristics of NMD substrates, the most prominent is structure specifically in the 3'UTR. We confirmed multiple NMD stability targets in sensory neurons including ATF4. AFT4 is preferentially translated during the integrated stress response (ISR). This led us to ask if suspension of NMD induces the ISR. Indeed, blockade of NMD increases eIF2-alpha phosphorylation, a key marker of the integrated stress response. Next, we examined the effects of SMG1 inhibition on pain-associated behaviors. Peripheral inhibition of SMG1 result in mechanical hypersensitivity that persists for several days and priming to a subthreshold dose of PGE2. Priming was fully rescued by a small molecule inhibitor of the ISR. Collectively, our results indicate that suspension of NMD promotes pain through activation of the ISR. Overall design: RNA-sequencing of mouse DRG cultures.

RNA稳定性受到精密调控。本研究旨在探究一种关键的转录后调控机制是否参与疼痛调控过程。无义介导的mRNA降解（Nonsense-mediated decay, NMD）可防止携带提前终止密码子的mRNA发生翻译，并调控约10%的典型蛋白编码mRNA的稳定性，该通路依赖于保守激酶SMG1的活性。SMG1及其靶标UPF1均在整个背根神经节（dorsal root ganglion, DRG）的感觉神经元中表达，坐骨神经中的DRG神经元及其神经纤维内亦可检测到SMG1蛋白。我们通过高通量测序技术，分析了SMG1抑制后的mRNA丰度变化。在NMD底物的诸多特征中，最显著的是其3'非翻译区（3' untranslated region, 3'UTR）的特定结构。我们在感觉神经元中验证了包括ATF4在内的多个NMD稳定性靶标。整合应激反应（integrated stress response, ISR）期间，ATF4会被优先翻译，这促使我们探究阻断NMD是否会激活整合应激反应。确实，阻断NMD可增加eIF2α的磷酸化水平，这是整合应激反应的关键标志物。随后，我们探究了SMG1抑制对疼痛相关行为的影响。外周给予SMG1抑制剂可引发持续数日的机械痛敏，并可使机体对阈下剂量的前列腺素E2（Prostaglandin E2, PGE2）产生致敏效应。整合应激反应的小分子抑制剂可完全逆转该致敏效应。综上，本研究结果表明，阻断NMD可通过激活整合应激反应促进疼痛发生。整体实验设计：小鼠DRG原代培养物的RNA测序。