责编 | 兮
细胞内的RNA上存在有大约150多种化学修饰，而N6-腺嘌呤A甲基化（N6-methyladenosine, m6A）是目前已知的真核生物mRNA修饰中最常见的一种【1】。自20世纪70年代被发现后的近四十年，m6A在细胞内的功能一直不是很清楚。近年来，随着高分辨率测序技术和高灵敏度质谱技术的发展，科学家逐渐揭开了m6A的神秘面纱。mRNA上的m6A由甲基化酶复合体（METTL3/METTL14）催化，并可以被去甲基化酶FTO和ALKBH5去除。m6A去甲基化酶的发现，证实了m6A是动态可调节的，进而将m6A研究推向了一个新的高潮【2】。m6A参与调节了mRNA代谢的所有阶段，从细胞核内的加工、出核到细胞质中的翻译和降解等；m6A也被证实调控多种生理病理过程。然而关于m6A如何被调节的分子机制目前尚不是特别明朗。
2021年1月18日，美国希望之城杨延钟研究组和费城儿童医院和宾夕法尼亚大学林兰研究组合作在EMBO Journal发表了题为 m6A deposition is regulated by PRMT1-mediated arginine methylation of METTL14 in its disordered C-terminal region 的研究论文【3】。该研究证实精氨酸甲基转移酶1（PRMT1）可以通过甲基化METTL14来调控m6A，并且证实了METTL14的精氨酸甲基化在细胞修复DNA链间交联的过程中起着非常重要的作用。
研究人员发现在METTL14的碳末端，有一个固有的蛋白无序区，这个固有的蛋白无序区具有非常多的RGG/RG重复序列， RGG/RG重复序列可以介导蛋白-蛋白和蛋白-核酸之间的相互作用。METTL14在m6A甲基化酶复合体的功能主要是负责RNA呈递，因此研究者测试了这个无序区对m6A甲基化酶复合体的功能的影响并证实了这个区域对于m6A甲基化酶复合体的功能是必须的。
拥有RGG/RG重复序列的蛋白大部分都是精氨酸甲基转移酶家族的底物。研究者通过体外反应发现PRMT1/PRMT3/PRMT6都可以在体外甲基化METTL14，而且甲基化就发生在这个固有的蛋白无序区，细胞内的免疫沉淀实验也证实了这一点。随后研究者证实了只有PRMT1可以在细胞内和METTL14相互作用并调控METTL14的精氨酸甲基化。
为了找到METTL14精氨酸甲基化修饰的功能，研究者使用了两个工具：一是I型精氨酸甲基转移酶的抑制剂MS023，它可以抑制PRMT1的活性从而抑制METTL14的甲基化；二是研究者通过质谱和体外甲基化反应找到了甲基化的位点并构建了甲基化缺失的突变型载体（METTL14 R→K, 将甲基化的精氨酸突变为赖氨酸）。通过这两个工具，研究者发现精氨酸甲基化缺失的m6A甲基化酶复合体的活性较野生型降低了近50%。考虑到METTL14在m6A甲基化酶复合体的功能主要是负责RNA呈递，研究者测试了野生型和甲基化缺失的METTL14蛋白与RNA底物的结合能力，发现METTL14的精氨酸甲基化对于METTL14和RNA底物的结合是非常重要的。此前的研究证实，mRNA上的m6A修饰是与转录共同发生的，METTL14与RNA 聚合酶II的相互作用在这个过程中起着非常重要的作用【4】。研究者发现METTL14的精氨酸甲基化对于其和RNA 聚合酶II的反应是必需的。
随后研究者通过慢病毒感染METTL14敲除的鼠胚胎干细胞，构建了过表达METTL14野生型和精氨酸甲基化缺失突变型的鼠胚胎干细胞系。和体外的生化反应结果一致，液相色谱-串联质谱法检测发现METTL14精氨酸甲基化缺失的鼠胚胎干细胞系m6A/A比例显著降低。接下来研究者通过m6A测序找到了METTL14精氨酸甲基化所调节的信号通路。以Fanconi Anemia信号通路为例，研究者发现METTL14的精氨酸甲基化可以通过调节这个信号通路里Atrip、Fancm和Palb2三个基因的m6A水平来调控它们的翻译效率，精氨酸甲基化缺失的鼠胚胎干细胞系里，Atrip、Fancm和Palb2的蛋白表达水平明显降低。Fanconi Anemia信号通路对于细胞修复DNA链间交联是必须的。研究者发现，相较于野生型, METTL14精氨酸甲基化缺失的细胞对Mitomycin C和顺铂造成的链间交联损伤异常敏感。
上述工作揭示，PRMT1可以通过精氨酸甲基化METTL14来调节其与RNA底物的结合，并调控METTL14与RNA聚合酶II的相互作用，进而提高细胞内的m6A水平。此外METTL14的精氨酸甲基化还可以通过调控Fanconi Anemia信号通路中关键基因的翻译效率来调节细胞对DNA链间交联的修复。
该研究由美国希望之城杨延钟教授和宾夕法尼亚大学费城儿童医院林兰教授领导的团队完成，该研究同时得到了美国芝加哥大学化学系何川教授的合作支持。博士后王志浩和博士生潘智城是本论文的共同第一作者。
杨延钟课题组招聘博士后
Postdoctoral Fellow – Epigenetics and RNA modification
The Beckman Research Institute of City of Hope is looking for a talented Postdoctoral Fellow in the laboratory of Dr. Yanzhong (Frankie) Yang at the Department of Cancer Genetics and Epigenetics. The research projects in Yang’s lab focus on investigating the role of protein arginine methylation in the regulation of epigenetics and RNA metabolism, particularly RNA modification and splicing. Arginine methylation is one of the most abundant protein modifications. Importantly, the enzymes that catalyze arginine methylation, namely protein arginine methyltransferases (PRMTs), are essential for development and have emerged as novel drug targets for cancer treatment. The lab utilizes biochemistry, molecular and structural biology, as well as cellular and genetically modified mouse models to address some of the most fundamental questions in the field of epigenetics and RNA metabolism, in which arginine methylation plays a critical role. To know more about Yang lab’s research, please refer to our recent publications (Mol Cell 40, 1016-1023 (2010); Mol Cell 53, 484-497 (2014); Nat Rev Cancer. 13, 37-50 (2013), and Nat Commun. 6:6428 (2015)).
The ideal candidate should have extensive experience in biochemistry, molecular biology, and cell biology in the field of epigenetics and RNA metabolism. Skills with mouse models are preferred. The candidate should be self-motivated, with a proven track record of productivity.
Basic education, experience and skills required for consideration:
- PhD in Biochemistry, Molecular Biology, and Cellular or Developmental Biology.
- Strong interest in basic and translational research.
- Strong work ethic, meticulous and detail-oriented record keeping habits.
- Excellent written and verbal communication skills. Evidence of productivity reflected in first author publications in peer-reviewed journals.
- Good problem solving skills and passion to follow relevant literature.
- Ability to conduct independent research, and work effectively in a team.
** Candidates that are interested can reach out directly to Dr. Yang at yyang@coh.org **
About City of Hope
City of Hope, an innovative biomedical research, treatment and educational institution with over 6000 employees, is dedicated to the prevention and cure of cancer and other life-threatening diseases and guided by a compassionate, patient-centered philosophy.
Founded in 1913 and headquartered in Duarte, California, City of Hope is a remarkable non-profit institution, where compassion and advanced care go hand-in-hand with excellence in clinical and scientific research. City of Hope is a National Cancer Institute designated Comprehensive Cancer Center and a founding member of the National Comprehensive Cancer Network, an alliance of the nation’s leading cancer centers that develops and institutes standards of care for cancer treatment.
原文链接：
https://www.embopress.org/doi/abs/10.15252/embj.2020106309
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