Alternative splicing of METTL3 explains apparently METTL3-independent m6A modifications in mRNA.
Alternative splicing of METTL3 explains apparently METTL3-independent m6A modifications in mRNA.
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METTL3的选择性剪接显然解释了mRNA中与METTL3无关的m6A修饰。
DOI：
10.1371/journal.pbio.3001683
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发表时间：
2022-07
期刊：
影响因子：
9.8
通讯作者：
中科院分区：
文献类型：
--
作者：
n6 -甲基腺苷（m6A）是一种非常普遍的mRNA修饰，可促进编码蛋白质的转录本降解，这些蛋白质在细胞发育、分化和其他途径中起作用。METTL3是催化mRNA中m6A形成的主要甲基转移酶。由于通过基于CRISPR/ cas9的方法，在METTL3耗尽后，30%至80%的m6A可以留在mRNA中，因此其他酶被认为可以催化相当一部分m6A。在这里，我们重新检查了mRNA转录组中m6A的来源。我们鉴定了在敲除Mettl3后其mRNA中继续含有m6A的小鼠胚胎干细胞系。我们发现这些细胞表达替代剪接的Mettl3转录异构体，绕过CRISPR/Cas9突变并产生功能活跃的甲基转移酶。我们类似地表明，其他报道的METTL3敲除细胞系表达改变的METTL3蛋白。我们发现基因依赖数据集显示，大多数细胞系在METTL3缺失后不能增殖，这表明报道的METTL3敲除细胞系表达改变的METTL3蛋白，而不是完全敲除。最后，我们利用METTL3的外显子4缺失重新评估了METTL3在m6A合成中的作用，发现METTL3负责mRNA中95%的m6A。总的来说，这些研究表明METTL3负责转录组中绝大多数m6A，并且在假定的METTL3敲除细胞系中剩余的m6A是由于改变但功能性的METTL3亚型的表达。在METTL3缺失后，修饰m6A仍然存在于mRNA中，这表明存在其他m6A甲基转移酶。本研究对METTL3基因敲除进行了研究，发现它们通常通过表达功能性METTL3亚型来逃避敲除，并证明METTL3确实负责mRNA中的大多数m6A。
N6-methyladenosine (m6A) is a highly prevalent mRNA modification that promotes degradation of transcripts encoding proteins that have roles in cell development, differentiation, and other pathways. METTL3 is the major methyltransferase that catalyzes the formation of m6A in mRNA. As 30% to 80% of m6A can remain in mRNA after METTL3 depletion by CRISPR/Cas9-based methods, other enzymes are thought to catalyze a sizable fraction of m6A. Here, we reexamined the source of m6A in the mRNA transcriptome. We characterized mouse embryonic stem cell lines that continue to have m6A in their mRNA after Mettl3 knockout. We show that these cells express alternatively spliced Mettl3 transcript isoforms that bypass the CRISPR/Cas9 mutations and produce functionally active methyltransferases. We similarly show that other reported METTL3 knockout cell lines express altered METTL3 proteins. We find that gene dependency datasets show that most cell lines fail to proliferate after METTL3 deletion, suggesting that reported METTL3 knockout cell lines express altered METTL3 proteins rather than have full knockout. Finally, we reassessed METTL3’s role in synthesizing m6A using an exon 4 deletion of Mettl3 and found that METTL3 is responsible for >95% of m6A in mRNA. Overall, these studies suggest that METTL3 is responsible for the vast majority of m6A in the transcriptome, and that remaining m6A in putative METTL3 knockout cell lines is due to the expression of altered but functional METTL3 isoforms. The modification m6A remains in mRNA after METTL3 depletion, suggesting that other m6A methyltransferases exist. This study investigates METTL3 knockouts, finding that they often escape knockout by expressing functional METTL3 hypomorphs, and demonstrating that METTL3 is indeed responsible for most m6A in mRNA.
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影响因子：
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作者：
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