Disrupted Minor Intron Splicing Activates Reductive Carboxylation-mediated Lipogenesis to Drive MASH Progression

Aberrant RNA splicing is tightly linked to diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD). Here, we revealed that minor intron splicing, a unique and conserved RNA processing event, is largely disrupted upon the progression of metabolic dysfunction-associated steatohepatitis (MASH) in mice and humans. We demonstrated deficiency of minor intron splicing in the liver induces MASH transition upon obesity-induced insulin resistance and LXR activation. Mechanistically, inactivation of minor intron splicing leads to minor intron retention of Insig1 and Insig2, resulting in premature termination of translation, which drives proteolytic activation of SREBP1c. This mechanism is conserved in human patients with MASH. Notably, disrupted minor intron splicing activates glutamine reductive metabolism for de novo lipogenesis through the induction of Idh1, which causes the accumulation of ammonia in the liver, thereby initiating hepatic fibrosis upon LXR activation. Ammonia clearance or IDH1 inhibition blocks hepatic fibrogenesis and mitigates MASH progression. More importantly, the overexpression of Zrsr1 restored minor intron retention and ameliorated the development of MASH, indicating that dysfunctional minor intron splicing is an emerging pathogenic mechanism that drives MASH progression. Additionally, reductive carboxylation flux triggered by minor intron retention in hepatocytes serves as a crucial checkpoint and potential target for MASH therapy. To investigate the gene expression in mouse liver regulated by Zrsr1/2, we established the Zrsr1/2 liver specific knock out mice by injection AAV expressing sgRNA targeting Zrsr1 and Zrsr2 (Zrsg) or AAV expressing GFP(control group) to Cas9 transgenic mouse through tail vein. After AAV injection ,all mice were maintained in 26℃ and fed with normal chow diet for 2 weeks, then were euthanasia and liver tissue were harvest and frozed immediately with liquid nitrogen. we performed gene expression profile using data obtained from RNA-seq of liver from control and Zrsg mice.

异常RNA剪接（aberrant RNA splicing）与多种疾病密切相关，其中包括代谢功能障碍相关脂肪性肝病（metabolic dysfunction-associated steatotic liver disease, MASLD）。本研究揭示，作为一类独特且保守的RNA加工事件，次要内含子剪接（minor intron splicing）在小鼠与人类的代谢功能障碍相关脂肪性肝炎（metabolic dysfunction-associated steatohepatitis, MASH）进展过程中受到广泛破坏。本研究证实，肝脏内次要内含子剪接缺陷可在肥胖诱导的胰岛素抵抗与肝X受体（liver X receptor, LXR）激活的背景下，诱发MASH的病程转变。
机制层面来看，次要内含子剪接失活会导致胰岛素诱导基因1（insulin-induced gene 1, Insig1）与胰岛素诱导基因2（insulin-induced gene 2, Insig2）出现次要内含子滞留，引发翻译提前终止，进而驱动固醇调节元件结合蛋白1c（sterol regulatory element-binding protein 1c, SREBP1c）的蛋白水解激活。该机制在人类MASH患者中同样保守存在。值得注意的是，破坏的次要内含子剪接可通过诱导异柠檬酸脱氢酶1（isocitrate dehydrogenase 1, IDH1）的表达，激活谷氨酰胺还原代谢以支持从头脂肪生成，这会导致肝脏内氨蓄积，最终在LXR激活的情况下引发肝纤维化（hepatic fibrosis）。清除氨或抑制IDH1可阻断肝纤维化进程并缓解MASH进展。
尤为重要的是，过表达Zrsr1可恢复次要内含子滞留现象并改善MASH的病程发展，这表明功能异常的次要内含子剪接是驱动MASH进展的新型致病机制。此外，肝细胞内由次要内含子滞留触发的还原性羧化通量可作为MASH治疗的关键检查点与潜在靶点。
为探究Zrsr1/2对小鼠肝脏基因表达的调控作用，我们通过尾静脉注射两种腺相关病毒（adeno-associated virus, AAV）至Cas9转基因小鼠体内：一种为携带靶向Zrsr1与Zrsr2的单向导RNA（single guide RNA, sgRNA）的AAV（Zrsg组），另一种为携带绿色荧光蛋白（green fluorescent protein, GFP）的对照AAV，以此构建Zrsr1/2肝脏特异性敲除小鼠模型。AAV注射完成后，所有小鼠于26℃环境下饲养并饲喂正常饲料2周，随后实施安乐死，迅速采集肝脏组织并置于液氮中速冻保存。我们通过对照小鼠与Zrsg小鼠的肝脏RNA测序（RNA-seq）数据开展了基因表达谱分析。