RNA m6A methylation contributes to hepatic inflammation in obesity-associated NAFLD - SPECIFIC AIMS: Obesity-induced metabolic inflammation results in the transition of nonalcoholic fatty liver disease (NAFLD) to steatohepatitis (NASH) [1-3]. Myeloid cell populations such as macrophages have been established as the main driver of metabolic inflammation [4-10]. Single-cell RNA sequencing (scRNA-seq) studies have provided a high-resolution fate map of myeloid diversity in NAFLD associated with distinct epigenomes, transcriptomes, and functions [9-11]. Recent advances emphasize the importance of post-transcriptional regulation of gene expression at the RNA level in obesity-driven hepatic inflammation [12, 13]. N6-methyladenosine (m6A) methylation represents the most prevalent and abundant epigenetic modification of eukaryotic mRNA. m6A methylation is dynamically installed, erased, and recognized by m6A methyltransferases (writers), m6A demethylases (erasers), and m6A specific binding enzymes (readers) [14, 15]. Highly m6A modified transcripts generally result in lower mRNA stability leading to reduced mRNA levels and thus greatly influence diverse cellular and biological responses [16-18]. The reader proteins recognize the m6A-modified sequences and determine the fate of the target mRNA. We recently identified that myeloid lineage-restricted deletion of m6A writer protein methyltransferase like 3 (METTL3) results in differential expression of m6A modified mRNA transcripts, particularly DNA Damage Inducible Transcript 4 (DDIT4) that negatively regulates mTOR/NFkB activation resulting in decreased immune cell infiltration, recovered liver homeostasis, improved hepatic metabolic abnormalities, and reduced inflammation during diet-induced obesity/NAFLD [19]. Although macrophage METTL3 plays an essential role in obesity-associated NAFLD, how it competes and collaborates with distinct m6A readers has not been identified, nor has the specificity of the methylation reaction and RNA structural motif determined. The molecular and cellular effects of m6A methylation in myeloid cell subsets, in addition to reducing mRNA stability, have yet to be elucidated in advanced fibrosis. DEAD-box RNA helicase DDX21 plays multifaceted roles in ribosome biogenesis, RNA editing, RNA transport, and transcription. DDX21 has been reported to interact with m6A writer proteins. Analysis of publicly available RNA-CLIP sequencing data indicates the overlap between DDX21 binding regions and m6A methylation sites on target mRNA molecules. Our preliminary results showed that mice with DDX21 deficiency in myeloid lineage, analogous to METTL3 deficiency, resulted in significant prevention of diet-induced steatohepatitis and fibrosis. The scRNA-seq analysis revealed two distinct myeloid populations, liver-resident (KC-H) macrophages and monocyte-derived macrophages (Mo-M), that have opposite proportional mobilization between chow and diet-fed mice. The lack of DDX21 restored the proportion of KC-H in diet-fed mice while reducing the proportion of Mo-M along with reduced inflammatory markers, indicating DDX21 plays a novel role in liver myeloid cells during obesity-associated NAFLD. Therefore, we hypothesize that mRNA m6A methylation in myeloid cell subsets, in association with DDX21, leads to hepatic inflammation during obesity-associated NAFLD (Fig.1). Aim 1 Establish the role of METTL3 in myeloid cell-driven hepatic inflammation during obesity-associated NAFLD. 1.1) Determine the contributions of METTL3 in KC-H and Mo-M subsets to hepatic inflammation. 1.1.1) Obtain the transcriptome-wide map of dynamic mRNA m6A at single-nucleotide resolution in METTL3-deficient KC-H and Mo-M cells during diet-induced NAFLD. 1.1.2). Identify the myeloid subset in which METTL3 deficiency results in protection from metabolic inflammation. This aim will identify the m6A pathway in myeloid subsets and downstream genes that contribute to hepatic inflammation during obesity-associated NAFLD. Aim 2 Determine the mechanistic relationship between the RNA helicase DDX21 a
