缺氧通过乳酸脱氢酶促进mRNA中6-甲基腺嘌呤修饰的作用及机制

N6-methyladenosine (m6A) is the most abundant mRNA post-translational modification in human. Although m6A modification plays critical roles in RNA biology, little is known about the function and mechanism of RNA m6A modification in response to signal pathways. Based on our previous studies (Cancer Research 2018), it was found that hypoxia enhances mRNA m6A modification through the inhibition of FTO activity by 2-hydroxyglutarate, which is converted from 2-ketoglutarate by FTO-bound LDHA. Since m6A modification has been reported to play important roles in RNA biology, it is hypothesized that hypoxia-driven LDHA promotes m6A modification, thereby achieving selective mRNA processing, translation and degradation. It is also hypothesized that m6A modification serves as a reversible mark to distinguish hypoxia-promoted transcripts from pre-existing messages, and result in the precisely modulation of gene transcription and translation. In this study, anti-m6A mRNA-IP and NGS (Next generation sequencing) will be applied to build the hypoxia-promoted m6A epigenetic map and identify the hypoxia-promoted m6A target genes. Q-PCR and FISH will be carried out to investigate the mechanisms of m6A-promoted regulation of target genes. Then overexpression and knockdown will be applied to verify the role of these target genes in cells. In addition, Cas9-mediated cell and mouse model will be built to unveil the molecular mechanisms of hypoxia-promoted m6A. We hope that this study could shed some light on the function and mechanisms of mRNA m6A modification in response to signal pathways.

6-甲基腺嘌呤m6A是人体mRNA最主要修饰，具控制RNA剪切翻译及肿瘤发生等重要功能，但肿瘤相关通路对其的作用和机制仍待研究。申请人在研究抗癌药调节核酸脱甲基的基础上（Cancer Research，2018），发现缺氧经乳酸脱氢酶催化2酮基戊二酸变成2羟基戊二酸2HG，且乳酸脱氢酶与m6A脱甲基酶FTO相结合，提高FTO周围局部2HG浓度，抑制FTO活性，进而增加m6A。据此，提出假说--缺氧经乳酸脱氢酶增加m6A，为新生成mRNA加上m6A标签，区分缺氧前后mRNA，以精确控制基因表达。拟用m6A抗体进行RNA沉淀结合测序，构建表达和m6A修饰图谱，鉴定缺氧经m6A调控的靶基因；以qPCR及原位杂交等技术，明确m6A调控靶基因的机制；以过表达和敲低，结合表型分析，研究靶基因功能；再以CAS9技术构建细胞和小鼠模型，验证相关机制，为阐明肿瘤相关通路对RNA修饰的作用和机制提供新思路。

N6-甲基腺嘌呤(N6-methyladenosine,m6A)是人体最主要的mRNA转录后修饰，在RNA的剪切、翻译和降解等过程中起到关键作用，是目前一大研究热点。但细胞对甲基腺嘌呤修饰的作用和调控机制尚知之甚少。同时，机体一般通过关键代谢酶的mRNA转录和翻译来调控代谢；而脱甲基关键酶fat mass and obesity associated（FTO）肥胖相关蛋白与代谢疾病高度相关，提示代谢酶或代谢物可通过FTO调控RNA m6A修饰。我们通过代谢物筛选结合质谱分析，发现乳酸脱氢酶LDHA（代谢酶）和NADPH（代谢物）可显著FTO酶活性，控制RNA m6A修饰，进而调控脂肪形成等多种生理病理过程。本项目的结果深化了代谢调控表观遗传学的认识，为阐明RNA甲基化修饰的作用和调控机制奠定基础，为寻找更有效的疾病治疗靶点提供新思路。