Role of hepatic Mettl14 pathways in liver metabolism and body metabolic homeostasis

信使RNA介导遗传信息翻译成蛋白质合成和细胞功能。具有免疫活性的mRNA的水平由RNA转录、转录后修饰和RNA与蛋白质的相互作用决定。转录的遗传和表观遗传调控已被广泛研究;相比之下，我们对mRNA修饰，RNA-蛋白质相互作用及其对细胞反应的影响的理解存在差距。N6-甲基腺苷（m6 A）是主要的mRNA修饰，并且由Mettl 3（催化亚基）/Mettl 14（结构亚基）甲基转移酶复合物催化。M6 A基序被Ythdc 1或相关RNA结合蛋白识别并结合，进而控制前mRNA剪接、核输出、细胞内定位、衰变和/或靶mRNA的翻译效率。Mettl 3、Mettl 14或Ythdc 1的全局缺失导致胚胎死亡，揭示了这些表转录组介质在发育和存活中的重要作用。肝脏是重要的代谢器官。肝病，包括非酒精性脂肪性肝病（NAFLD）和代谢功能障碍，是死亡和发病的主要原因。然而，基于Mettl 14/Ythdc 1的epitranscriptomic程序尚未在肝脏中探索。在初步研究中，我们产生了成年发病，肝细胞特异性Mettl 14和Ythdc 1基因敲除小鼠。肝Mettl 14的消融减弱了高脂饮食（HFD）诱导的高血糖症、葡萄糖耐受不良和肝脂肪变性。同样，肝细胞特异性缺失Ythdc 1也改善了HFD喂养小鼠的葡萄糖代谢。在肝切片培养中，Mettl 14缺乏减弱胰高血糖素刺激的肝脏葡萄糖产生。RNA-seq分析表明，肝Mettl 14的缺失改变了许多肝脏mRNA转录物的水平，这些转录物编码参与葡萄糖/脂质代谢和信号传导途径的蛋白质。基于这些结果，我们假设Mettl 14介导编码肝脏糖异生、脂肪生成和相关信号通路的介导剂/调节剂的mRNA中的m6 A甲基化。Ythdc 1与m6 A基序结合，并控制前mRNA剪接、核输出和/或Mettl 14底物亚组的降解。此外，肥胖相关因素增加肝脏Mettl 14和Ythdc 1的表达和稳定性，从而引发基于Mettl 14/Ythdc 1的肝脏代谢表观重编程。Mettl 14/m6 A/Ythdc 1诱导的表观转录组重编程提供了肥胖相关NAFLD和2型糖尿病的新机制。目的1确定肝Mettl 14是否通过表转录组学机制直接促进肝脏葡萄糖产生和肝脏脂肪变性。目的2确定Ythdc 1是否介导Mettl 14在肝脏中的代谢作用。目的3确定Mettl 14/Ythdc 1诱导的表位转录组重编程是否介导肥胖相关的肝病。该项目的成果有望建立一种新的基于Mett 14/Ythdc 1的表转录组重编程范式，用于管理健康和疾病中的肝脏代谢。

Messenger RNA mediates translation of genetic information into protein synthesis and cell functions. Levels of translationally-competent mRNA are determined by RNA transcription, post-transcriptional modifications, and RNA interactions with proteins. Genetic and epigenetic regulations of transcription have been extensively investigated; by contrast, there is a gap in our understanding of mRNA modifications, RNA-protein interactions, and their impacts on cellular responses. N6-methyladenosine (m6A) is the predominant mRNA modification, and is catalyzed by a Mettl3 (catalytic subunit)/Mettl14 (structural subunit) methyltransferase complex. M6A motif is recognized by and bound to Ythdc1 or related RNA-binding proteins, which in turn control pre-mRNA splicing, nuclear export, intracellular localization, decay, and/or translational efficiency of target mRNAs. Global deletion of Mettl3, Mettl14, or Ythdc1 results in embryonic death, revealing the essential role of these epitranscriptomic mediators in development and survival. Liver is an essential metabolic organ. Liver disease, including nonalcoholic fatty liver disease (NAFLD) and metabolic dysfunctions, is a main cause for mortality and morbidity. However, Mettl14/Ythdc1-based epitranscriptomic programs have not been explored in the liver. In the preliminary study, we generated adult-onset, hepatocyte-specific Mettl14 and Ythdc1 knockout mice. Ablation of hepatic Mettl14 attenuated high fat diet (HFD)-induced hyperglycemia, glucose intolerance, and liver steatosis. Likewise, hepatocyte-specific deletion of Ythdc1 also improved glucose metabolism in HFD-fed mice. In liver slice cultures, Mettl14 deficiency blunted glucagon-stimulated liver glucose production. RNA-seq analysis showed that deletion of hepatic Mettl14 changed levels of many liver mRNA transcripts encoding proteins involved in glucose/lipid metabolism and signaling pathways. Based on these results, we hypothesize that Mettl14 mediates m6A methylation in mRNAs encoding mediators/modulates for hepatic gluconeogenesis, lipogenesis, and related signaling pathways. Ythdc1 binds to m6A motif, and controls pre-mRNA splicing, nuclear export, and/or degradation of a subset of Mettl14 substrates. Moreover, obesity-related factors increase expression and stability of hepatic Mettl14 and Ythdc1, thereby eliciting Mettl14/Ythdc1-based epitranscriptomic reprogramming of liver metabolism. Mettl14/m6A/Ythdc1-elicited epitranscriptomic reprogramming provides a new mechanism underlying obesity-associated NAFLD and type 2 diabetes. Aim 1 determines whether hepatic Mettl14 directly promotes liver glucose production and liver steatosis by an epitranscriptomic mechanism. Aim 2 determines whether Ythdc1 mediates the metabolic action of Mettl14 in the liver. Aim 3 determines whether Mettl14/Ythdc1-elicited epitranscriptomic reprogramming mediates obesity- associated liver disease. The outcomes of this project are expected to establish a new Mett14/Ythdc1-based epitranscriptomic reprogramming paradigm governing liver metabolism in health and disease.