Determine the methyltransferase activities of METTL16 via MeRIP-seq (m6A-seq)

METTL16 belongs to methyltransferase like (METTL) family and could install m6A marks on its substrates. Here, we uncover a tumor-promoting role of METTL16 in AML and LSC self-renewal. To explore the mechanism underlying the oncogenic function of METTL16 in AML, we performed m6A-seq and RNA-seq. Via integrated analysis of m6A-seq data and RNA-seq data, we identified two bona fide targets of METTL16, BCAT1 and BCAT2. METTL16 functions as an m6A methyltransferase to regulate expression of BCAT1 and BCAT2, which contribute to reprogramming of BCAA metabolism. The NOMO-1 cells were infected with sgMETTL16-1, sgMETTL16-2,and sgNS lentivirus to induce knockout of METTL16. The positive-infected cells were selected with 2ug/ml puromycin for 1-2 weeks. The cells were collected and total RNA were isolated from the whole cells via Qiazol. The poly(A) RNAs were further enriched from the total RNA and then subjected to m6A IP.

METTL16属于甲基转移酶样（methyltransferase like, METTL）家族，可在其底物上催化引入N6-甲基腺苷（m6A）修饰。本研究揭示了METTL16在急性髓系白血病（Acute Myeloid Leukemia, AML）及白血病干细胞（Leukemia Stem Cells, LSC）自我更新中的促肿瘤作用。为探究METTL16在急性髓系白血病中发挥致癌功能的潜在分子机制，我们开展了m6A测序（m6A-seq）与RNA测序（RNA-seq）实验。通过对m6A测序数据及RNA测序数据的整合分析，我们鉴定出METTL16的两个确证靶点：BCAT1与BCAT2。METTL16作为m6A甲基转移酶，可调控BCAT1与BCAT2的表达，进而参与支链氨基酸（Branched-Chain Amino Acid, BCAA）代谢重编程。我们使用sgMETTL16-1、sgMETTL16-2及sgNS慢病毒感染NOMO-1细胞，以实现METTL16基因的敲除。随后用2μg/ml嘌呤霉素（puromycin）筛选感染阳性的细胞，筛选时长为1-2周。收集细胞后，通过Qiazol试剂从全细胞中提取总RNA。从总RNA中进一步富集聚腺苷酸化RNA（poly(A) RNA），随后进行m6A免疫沉淀（m6A IP）实验。