人源m^(6)A甲基转移酶复合物METTL3-METTL14的表达、纯化和透射电镜观察

Expression,Purification and TEM Observation of m^(6)A Methyltransferase Complex METTL3-METTL14

N^(6)-腺苷酸甲基化(m^(6)A)是一种重要的mRNA和长链非编码RNA的内部修饰方式,通过影响RNA代谢的各个方面,参与调控人体一系列生理过程。METTL3和METTL14组成的甲基转移酶复合物可以有效地催化甲基基团的转移,但其具体的催化机制尚不完全清楚。本研究通过进行蛋白质结构无序度分析及三维模型预测发现,二者除MTD结构域外其他区域基本处于无序或低复杂度状态。为了进一步探索METTL3-METTL14复合物实现甲基基团转移的结构基础,本研究在哺乳动物细胞HEK293S GnTI-中成功表达了全长序列的人源METTL3-METTL14蛋白复合物,并通过镍柱亲和层析、离子交换及凝胶过滤层析获得了高纯度的METTL3-METTL14蛋白复合物,并推测全长METTL3和METTL14以1∶1的比例组成复合物。通过120kV透射电子显微镜观察发现,蛋白颗粒大小均一,颗粒直径在10~12nm。

N^(6)-adenylate methylation(m^(6)A)is an important internal modification of mRNA and long non-coding RNAs,which is involved in regulating a series of physiological processes by affecting various aspects of RNA metabolism.The methyltransferase complex composed of METTL3 and METTL14 can effectively catalyze the transfer of methyl groups,but the specific catalytic mechanism is not completely clear.In this study,protein sequence analysis and 3D model prediction suggested that the two proteins contain disordered regions outside of the MTD region.In order to further explore the structural basis of METTL3-METTL14 complex for methyl group transfer,the full-length human METTL3-METTL14 protein complex was successfully expressed in mammalian cell HEK293S GnTI-,and high purity METTL3-METTL14 protein complex was obtained by sequential purification using Ni-column affinity chromatography,ion exchange chromatography and gel filtration chromatography.It was likely that the full-length METTL3 and METTL14 formed a complex at a 1∶1 ratio.Using 120kV transmission electron microscope,protein particles were found to have diameters of 10—12nm.