RIP-seq analysis of Usp39 binding sites in mouse AML12 cell line

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. RNA immunoprecipitation sequencing (RIP-seq) for splicing factor Usp39 in mouse AML12 cells.