《植物生理学报》 2021, 57(3): 605-613

拟南芥ECT7蛋白的相互作用蛋白及翻译后修饰的鉴定

李阳晨，程玲，卞江虎，王文斐^*

福建农林大学生命科学学院, 福州350002

通信作者：王文斐；E-mail: wenfeiwang@fafu.edu.cn

摘　要：

Evolutionarily conserved C-terminal region (ECT)蛋白是植物中的一类RNA结合蛋白, 负责识别RNA的6-甲基腺嘌呤(N⁶-methyl-adenosine, m⁶A)修饰。 m⁶A结合蛋白被报道在动物体的生命活动及发育过程中起重要调控作用, 而植物中ECT家族仅有少量报道且具体功能及工作机制尚不清楚, 亟待研究。本文利用基于稳定同位素标记的定量蛋白质组学方法鉴定了ECT7蛋白的相互作用蛋白及翻译后修饰, 共鉴定到45个ECT7的相互作用蛋白, 其中包括其自身和同源蛋白ECT8 (evolutionarily conserved C-terminal region 8)在内的5个RNA结合蛋白, 并利用酵母双杂交实验对ECT家族蛋白之间进行了互作验证, 发现ECT7可与多个ECT家族成员相互作用。不仅如此, 还鉴定到ECT7蛋白有4个氧连-氮乙酰葡萄糖胺(O-linked β-N-acetyl glucosamine, O-GlcNAc)修饰位点和4个磷酸化修饰位点。研究结果对于进一步研究ECT7蛋白的功能及其识别m⁶A修饰的分子机制提供了理论依据。

关键词：拟南芥; ECT7; m6A读码器; RNA结合蛋白; 基于同位素标记的定量免疫沉淀-质谱联用(SILIP-MS)

收稿：2020-10-22   修定：2021-01-07

资助：国家自然科学基金(31700254)

Identifcation of ECT7-interacting proteins and post-translational modifcations of ECT7 protein in Arabidopsis

LI Yangchen, CHENG Ling, BIAN Jianghu, WANG Wenfe^*

College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China

Corresponding author: WANG Wenfe; E-mail: wenfeiwang@fafu.edu.cn

Abstract:

Evolutionarily conserved C-terminal region (ECT) proteins are a kind of RNA binding proteins, recognizing 6-methyl-adenosine (N⁶-methyl-adenosine, m⁶A) modifcation on RNA as reader proteins. In animals, m6A-binding proteins have been found to play important roles in multiple growth and developmental processes. However, only few studies of ECT family gene have been reported in plants. The function and working mechanism of ECT proteins are still unclear. Here, we performed stable isotope labeling immunoprecipitation followed by Mass Spectrometry (SILIP-MS) to investigate ECT7-interacting proteins and post-translational modification. 45 putative interacting proteins were found as ECT7 interacting proteins, including itself and its
homolog ECT8 (evolutionarily conserved C-terminal region 8) and other three RNA binding proteins. Furthermore, ECT7 was confrmed to interact with multiple ECT members by yeast two-hybrid experiment. In addition, we identifed 4 O-linked β-N-acetyl glucosamine (O-GlcNAc) modifcation sites and 4 phosphorylation sites on ECT7 protein. Our results provide theoretical basis for further research on the function of ECT7 protein and molecular mechanism of recognition m⁶A modifcation in plants.

Key words: Arabidopsis; ECT7; m6A reader; RNA-binding protein; quantitative stable isotope labeling Immunoprecipitation and Mass Spectrometry (SILIP-MS)