RNA modifications in apolipoprotein regulation by environmental exposures

Exposure to persistent environmental contaminants including polychlorinated biphenyls (PCBs) contributes to metabolic diseases including metabolic dysfunction-associated steatotic liver disease (MASLD). Although PCB exposures are positively associated with altered liver enzymes, miRNAs, MASLD, and mortality in human cohorts, how PCBs act in collaboration with a high fat diet (HFD) to promote increased hepatic triglyceride accumulation, fibrosis, and inflammation in progression to metabolic-dysfunction associated steatohepatitis (MASH) remains unknown. Further, although liver sexual dimorphism and sex-specific differences in human health outcomes after PCB exposure have been reported, few mechanistic studies have addressed these differences. We reported that livers from HFD-fed male mice with MASH after a single oral exposure to an environmentally-relevant mixture of non-dioxin-like PCBs, Aroclor1260 (Ar1260), and the dioxin-like PCB126 showed increased N(6)-methyladenosine (m6A), the most common RNA modification, that regulates transcript stability and alternative splicing (AS). We identified m6A in Apob (apolipoprotein b) and other MASLD-related transcripts using m6A mRNA immunoprecipitation (RIP)-sequencing and confirmed select m6A-differentially expressed genes using Nanopore direct RNA-seq. in AML12 mouse hepatocytes. Reduced APOB protein is critical for hepatic lipid accumulation in human MASLD. Apob is constitutively transcribed, suggesting that post- transcriptional regulation and protein stability determine APOB levels. These data support our overarching hypothesis that PCB exposures and diet alter m6A to regulate lipid metabolism pathways in MASLD. We address a critical knowledge gap in how diet, environmental pollutant exposures, and sex modify the m6A epitranscriptome to regulate apolipoprotein homeostasis. We will 1) Determine if the altered m6A marks in the Apob transcript with PCB exposures regulate transcript stability and translation. 2) Determine how sex and diet impact m6A marks in lipoprotein transcripts in a high fat, high fructose diet (HFHFD) + PCB exposure model of MASH. 3) Identify the extent of m6A-induced alternative splicing (AS) in livers in HFHFD-fed mice with PCB exposures. The resulting data will be the first to directly examine how mA6-containing Apob regulates protein abundance affecting hepatic lipid pathology. Determining the mechanistic interplay of m6A, diet, and PCB exposures in promoting NAFLD progression will reveal possible targets for therapeutic intervention. Project Number: 1R01ES036210-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Environmental Health Sciences (NIEHS) | Principal Investigator: Carolyn Klinge (+1 co-PI) | Institution: UNIVERSITY OF LOUISVILLE, LOUISVILLE, KY | Award Amount: $3,051,769 | Activity Code: R01 | Study Section: Environmental Determinants of Disease Study Section [EDD] View on NIH RePORTER: https://reporter.nih.gov/project-details/11225187