RNA sequencing of liver tissues from liver-specific Usp39 knockout (Usp39-HKO) mice

Regulation of alternative splicing (AS) is crucial for gene expression and enables a single transcript to yield multiple isoforms that increase transcriptome and proteome diversity. Dysregulated AS has been linked to the development of non-alcoholic fatty liver diseases (NAFLD). However, the splicing factors involved in hepatic homeostasis and their functional mechanisms remain to be further characterized. Here, we report that spliceosome component Usp39 plays a critical role in the regulation of hepatocyte lipid homeostasis. We found that Usp39 expression is downregulated in hepatic tissues of NAFLD and non-alcoholic steatohepatitis (NASH) subjects. We observed increased lipid accumulation, spontaneous steatosis and impaired autophagy, lipophagy in particular, in mice with hepatocyte-specific Usp39 deletion. Combined analysis of RIP-seq and RNA-seq data revealed that Usp39 regulates AS of several autophagy-related genes including Hsf1. More specifically, deletion of Usp39 resulted in alternative 5’ splice site selection of exon 6 in Hsf1 and consequently reduced expression. Hsf1 was also found to be downregulated in NAFLD/NASH mice and patients. Importantly, overexpression of Hsf1 restored lipophagy, attenuated lipid accumulation and alleviated NASH caused by Usp39 deficiency. Taken together, our findings indicate that Usp39-mediated AS is crucial for sustaining lipophagy and lipid homeostasis in the liver. Comparative gene expression profiling analysis of RNA-seq data for 5-week-old Usp39-HKO and control littermates liver

可变剪接(Alternative Splicing, AS)的调控对于基因表达至关重要，它可使单个转录本产生多种剪接异构体，进而增加转录组与蛋白质组的多样性。异常调控的可变剪接与非酒精性脂肪性肝病(Non-Alcoholic Fatty Liver Diseases, NAFLD)的发生发展密切相关。然而，参与肝脏稳态调控的剪接因子及其作用机制仍有待进一步阐明。本研究发现，剪接体组分Usp39在肝细胞脂质稳态调控中发挥关键作用。我们观察到，在非酒精性脂肪性肝病及非酒精性脂肪性肝炎(Non-Alcoholic Steatohepatitis, NASH)患者的肝组织中，Usp39的表达水平显著下调。在肝细胞特异性敲除Usp39的小鼠中，我们观察到脂质积累增多、自发性脂肪变性，且自噬功能受损，尤以脂噬为甚。通过对RNA免疫沉淀测序(RNA Immunoprecipitation Sequencing, RIP-seq)与转录组测序(RNA-sequencing, RNA-seq)数据的联合分析，我们发现Usp39可调控包括热休克因子1(Heat Shock Factor 1, Hsf1)在内的多个自噬相关基因的可变剪接。更为具体地，敲除Usp39会导致Hsf1第6外显子的可变5'剪接位点选择发生异常，最终使其表达水平下调。研究同时发现，在非酒精性脂肪性肝病/非酒精性脂肪性肝炎小鼠模型及患者体内，Hsf1的表达水平同样出现下调。重要的是，过表达Hsf1可恢复脂噬功能、减轻脂质积累，并缓解Usp39缺失所诱导的非酒精性脂肪性肝炎。综上，我们的研究结果表明，Usp39介导的可变剪接对于维持肝脏脂噬功能与脂质稳态至关重要。对5周龄Usp39肝细胞特异性敲除(Usp39-HKO)小鼠与同窝对照小鼠的肝组织转录组测序数据开展的比较基因表达谱分析