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

信使RNA介导遗传信息转化为蛋白质合成和细胞功能。翻译能力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 -蛋白相互作用及其对细胞反应的影响的理解存在空白。n6 -甲基腺苷（m6A）是主要的mRNA修饰，由Mettl3（催化亚基）/Mettl14（结构亚基）甲基转移酶复合物催化。M6A基序被Ythdc1或相关rna结合蛋白识别并结合，进而控制mrna前剪接、核输出、细胞内定位、衰变和/或靶mrna的翻译效率。Mettl3、Mettl14或Ythdc1的整体缺失导致胚胎死亡，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。肝脏疾病，包括非酒精性脂肪性肝病（NAFLD）和代谢功能障碍，是死亡率和发病率的主要原因。然而，基于Mettl14/ ythdc1的表转录组学程序尚未在肝脏中进行探索。在初步研究中，我们培育了成年发病的肝细胞特异性Mettl14和Ythdc1敲除小鼠。消融肝Mettl14可减轻高脂饮食（HFD）引起的高血糖、葡萄糖耐受不良和肝脂肪变性。同样，肝细胞特异性缺失Ythdc1也改善了hfd喂养小鼠的葡萄糖代谢。在肝切片培养中，缺乏Mettl14使胰高血糖素刺激的肝脏葡萄糖生成变钝。RNA-seq分析显示，肝脏Mettl14的缺失改变了许多肝脏mRNA转录物的水平，这些mRNA转录物编码了参与糖/脂代谢和信号通路的蛋白质。基于这些结果，我们假设Mettl14介导m6A甲基化的mrna编码介质/调节肝脏糖异生、脂肪生成和相关信号通路。Ythdc1结合m6A基序，控制前mrna剪接、核输出和/或Mettl14底物子集的降解。此外，肥胖相关因素增加了肝脏Mettl14和Ythdc1的表达和稳定性，从而引发基于Mettl14/Ythdc1的肝脏代谢表转录组重编程。Mettl14/m6A/ ythdc1引发的表转录组重编程为肥胖相关NAFLD和2型糖尿病提供了新的机制。目的1确定肝脏Mettl14是否通过表转录组学机制直接促进肝脏葡萄糖生成和肝脏脂肪变性。目的2确定Ythdc1是否介导肝脏中Mettl14的代谢作用。目的3确定Mettl14/ ythdc1引发的表转录组重编程是否介导肥胖相关的肝脏疾病。该项目的结果有望建立一种新的基于Mett14/ ythdc1的表转录组重编程范式，控制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.