From the ‡School of Biotechnology and Biomolecular Sciences, University of New South Wales, New South Wales, 2052, Australia.
From the ‡School of Biotechnology and Biomolecular Sciences, University of New South Wales, New South Wales, 2052, Australia
Mol Cell Proteomics. 2017 Dec;16(12):2229-2242. doi: 10.1074/mcp.M116.066308. Epub 2017 Jun 29.
Lysine methylation is widespread on human proteins, however the enzymes that catalyze its addition remain largely unknown. This limits our capacity to study the function and regulation of this modification. Here we used the CRISPR/Cas9 system to knockout putative protein methyltransferases and in K562 cells, to determine if they methylate elongation factor eEF1A. The known eEF1A methyltransferase was also knocked out as a control. Targeted mass spectrometry revealed the loss of lysine 165 methylation upon knockout of , and the expected loss of lysine 79 methylation on knockout of No loss of eEF1A methylation was seen in the knockout. Recombinant METTL21B was shown to catalyze methylation on lysine 165 in eEF1A1 and eEF1A2, confirming it as the methyltransferase responsible for this methylation site. Proteomic analysis by SILAC revealed specific upregulation of large ribosomal subunit proteins in the knockout, and changes to further processes related to eEF1A function in knockouts of both and This indicates that the methylation of lysine 165 in human eEF1A has a very specific role. METTL21B exists only in vertebrates, with its target lysine showing similar evolutionary conservation. We suggest METTL21B be renamed eEF1A-KMT3. This is the first study to specifically generate CRISPR/Cas9 knockouts of putative protein methyltransferase genes, for substrate discovery and site mapping. Our approach should prove useful for the discovery of further novel methyltransferases, and more generally for the discovery of sites for other protein-modifying enzymes.
赖氨酸甲基化广泛存在于人类蛋白质中，然而，催化其添加的酶仍然很大程度上未知。这限制了我们研究这种修饰的功能和调节的能力。在这里，我们使用 CRISPR/Cas9 系统敲除 K562 细胞中假定的蛋白甲基转移酶和，以确定它们是否甲基化延伸因子 eEF1A。作为对照，也敲除了已知的 eEF1A 甲基转移酶。靶向质谱分析显示，在敲除后赖氨酸 165 甲基化的丧失，以及在敲除后赖氨酸 79 甲基化的预期丧失。在 敲除中未观察到 eEF1A 甲基化的丢失。重组 METTL21B 被证明可以催化 eEF1A1 和 eEF1A2 上赖氨酸 165 的甲基化，证实它是负责该甲基化位点的甲基转移酶。SILAC 蛋白质组学分析显示，在 敲除中，核糖体大亚基蛋白特异性上调，并且在敲除和 中，与 eEF1A 功能相关的进一步过程发生变化。这表明人类 eEF1A 中赖氨酸 165 的甲基化具有非常特定的作用。METTL21B 仅存在于脊椎动物中，其靶赖氨酸显示出类似的进化保守性。我们建议将 METTL21B 重新命名为 eEF1A-KMT3。这是第一项专门针对潜在蛋白甲基转移酶基因进行 CRISPR/Cas9 敲除以进行底物发现和位点映射的研究。我们的方法应该有助于发现更多的新型甲基转移酶，更普遍地有助于发现其他蛋白修饰酶的位点。
