METTL3 alters capping enzyme expression and its activity on ribosomal proteins in NSCLC cell model. METTL3 alters capping enzyme expression and its activity on ribosomal proteins in NSCLC cell model

The 5’ cap, catalyzed by capping enzyme, RNGTT, is a vital mRNA modification for the functionality of mRNAs and dysregulated in cancer cells. The m6A writer METTL3 is a regulator of mRNA stability and protein translation, and its expression is altered in various cancers, including lung cancer cells. We identified a METTL3 motif in the mRNA of RNGTT and investigated whether METTL3 could regulate the expression of RNGTT. Knock down of METTL3 resulted in destabilizing RNGTT mRNA, reduced protein expression and activity. Sequencing of capped mRNAs and m7G immunoprecipitation identified an underrepresentation of ribosomal protein mRNA. Overall design: To investigate the regulatory relationship between METTL3 and RNGTT, we generated a stable cell line of A549 expressing either an sh- control or shMETTL3 We then performed a cDNA enrichment of capped mRNAs in biological triplicate of A549sh- and A549 shMETTL3. The then performed gene expression profile analysis using data obtained from DNAseq with the resulting cDNA.

由加帽酶RNGTT催化形成的5'帽结构（5' cap）是维系mRNA功能的关键转录后修饰类型，该修饰在癌细胞中存在调控异常。m6A甲基化写入酶（m6A writer）METTL3是调控mRNA稳定性与蛋白质翻译过程的核心因子，其表达水平在包括肺癌细胞在内的多种癌症中均发生异常改变。我们在RNGTT的mRNA序列中鉴定到了METTL3结合基序，并据此探究了METTL3对RNGTT基因表达的调控作用。敲低METTL3会导致RNGTT mRNA的稳定性下降，同时降低其蛋白质表达水平与酶活性。通过对帽结构mRNA进行测序以及m7G免疫沉淀（m7G immunoprecipitation）实验，我们发现核糖体蛋白mRNA的富集程度显著低于预期。整体实验设计：为探究METTL3与RNGTT之间的调控关联，我们构建了稳定表达短发夹RNA对照（sh- control）或shMETTL3的A549细胞系。随后，我们分别对A549sh-与A549shMETTL3细胞开展了三次生物学重复的帽结构mRNA cDNA富集实验，并基于获取的cDNA通过DNA测序（DNAseq）技术完成了基因表达谱分析。