Cancer mutations rewire the RNA methylation specificity of METTL3-METTL14

Chemical modification of RNAs is important for post-transcriptional gene regulation. The METTL3-METTL14 complex generates most N6-methyladenosine (m6A) modifications in mRNAs, and dysregulated methyltransferase expression has been linked to cancers. Here we show that a changed sequence context for m6A can promote oncogenesis. A gain-of-function missense mutation from cancer patients, METTL14R298P, increases malignant cell growth in culture and transgenic mice, without increasing global m6A levels in mRNAs. The mutant methyltransferase preferentially modifies noncanonical sites containing a GGAU motif, in vitro and in vivo. The m6A in GGAU context is detected by the YTH family of readers similarly to the canonical sites but is demethylated less efficiently by an eraser, ALKBH5. Combining the biochemical and structural data we provide a model for how the cognate RNA sequences are selected for methylation by METTL3-METTL14. Our work highlights that sequence-specific m6A deposition is impor..., In vitro methylation sequencing (IVM-seq)
A degenerate DNA oligonucleotide mixture (5'-CTCCTTCTGGCATAAGAAGTNNNNNNNNNNNNNNNNNNNNCAAGCCAAGCAAGTATATAGG-3') consisting of 20-random-nucleotide (N20) and flanking constant regions was synthesized by MilliporeSigma with manual adjustment to achieve the overall equal ratio of nucleotides. An initial double-stranded (ds)DNA library was produced by a 7-cycle PCR amplification (F-primer: 5'-ATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGCTCCTTCTGGCATAAGAAGT-3'; R-primer: 5'-CCTATATACTTGCTTGGCTTG-3') in a 1 mL reaction containing 20 pmol of N20 oligonucleotides as a template to preserve the designed library complexity. The dsDNA was then gel-eluted and transcribed into a randomized RNA library. The RNA library (6.25 µM) was methylated by the target recombinant methyltransferase heterodimer (0.25 µM) in a 20 µL reaction (50 mM Tris pH 7.5, 0.01% Triton X-100, 15 mM NaCl, 1 mM DTT, 1% glycerol, 5 µM S-(5′-adenosyl)-L-methionine (SAM) (Sigma A7007), 20 U SUPERa..., , # Cancer mutations rewire the RNA methylation specificity of METTL3-METTL14

[https://doi.org/10.5061/dryad.37pvmcvvb](https://doi.org/10.5061/dryad.37pvmcvvb)

## Description of the data and file structure

Raw sequencing data (.fastq.gz and .md5 files are compressed in each .zip file) include IVM-seq (abcam or sysy antibody) and SELEX experiments. The files with name containing \"ivm-seq\" or \"selex\" were derived from the corresponding experiment respectively. \"ivm_seq_sysy\" or \"ivm_seq_abcam\" indicates the parallel experiments performed by using sysy or abcam antibodies respectively. \"mock, wt, r298p, r298c, r298h, d312a\" indicates the experiment were performed by using no protein (mock pull-down control), wild-type METTL14 recombinant protein or corresponding METTL14 mutant protein. The details of raw sequencing data processing is included in the method section.Â

RNA的化学修饰在转录后基因调控中具有重要意义。METTL3-METTL14复合物是mRNA中大部分N6-甲基腺苷（m6A）修饰的产生者，而甲基转移酶表达失调与癌症相关。本研究表明，m6A的序列背景改变可促进肿瘤发生。来自癌症患者的功能获得性错义突变METTL14R298P可促进培养细胞和转基因小鼠中恶性细胞的生长，且不升高mRNA中的整体m6A水平。该突变型甲基转移酶在体外和体内均优先修饰含GGAU基序的非经典位点。GGAU背景下的m6A可被YTH家族的阅读器识别（与经典位点类似），但被去甲基化酶ALKBH5去甲基化的效率较低。结合生化和结构数据，我们提出了METTL3-METTL14选择同源RNA序列进行甲基化的模型。本研究强调序列特异性m6A沉积的重要性……体外甲基化测序（IVM-seq）

由MilliporeSigma合成了包含20个随机核苷酸（N20）和侧翼恒定区的简并DNA寡核苷酸混合物（序列：5'-CTCCTTCTGGCATAAGAAGTNNNNNNNNNNNNNNNNNNNNCAAGCCAAGCAAGTATATAGG-3'），并通过手动调整实现核苷酸的整体等比例分布。以20 pmol N20寡核苷酸为模板，通过7个循环的PCR扩增（正向引物：5'-ATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGCTCCTTCTGGCATAAGAAGT-3'；反向引物：5'-CCTATATACTTGCTTGGCTTG-3'）在1 mL反应体系中制备初始双链（ds）DNA文库，以保留设计的文库复杂度。随后将该dsDNA进行凝胶洗脱并转录为随机RNA文库。RNA文库（6.25 µM）在20 µL反应体系中（含50 mM Tris pH7.5、0.01% Triton X-100、15 mM NaCl、1 mM DTT、1%甘油、5 µM S-腺苷甲硫氨酸（SAM，Sigma A7007）、20 U SUPERa……）被目标重组甲基转移酶异二聚体（0.25 µM）甲基化。#癌症突变改变METTL3-METTL14的RNA甲基化特异性

[https://doi.org/10.5061/dryad.37pvmcvvb](https://doi.org/10.5061/dryad.37pvmcvvb)

##数据与文件结构描述

原始测序数据（每个.zip文件中压缩有.fastq.gz和.md5文件）包括IVM-seq（使用abcam或sysy抗体）和SELEX实验数据。文件名含"ivm-seq"或"selex"的文件分别来自对应实验。"ivm_seq_sysy"或"ivm_seq_abcam"表示分别使用sysy或abcam抗体进行的平行实验。"mock、wt、r298p、r298c、r298h、d312a"表示实验使用无蛋白（mock下拉对照）、野生型METTL14重组蛋白或相应METTL14突变体蛋白。原始测序数据处理的详细信息见方法部分。