Interactome of GIGYF2 and EIF4E2 with and without SMG1i treatement

Translation of messenger RNAs (mRNAs) with premature translation termination codons produces truncated proteins with potentially deleterious effects. This is prevented by nonsense-mediated mRNA decay (NMD) of these mRNAs. NMD is triggered by ribosomes terminating upstream of a splice site marked by an exon-junction complex (EJC), but also acts on many mRNAs lacking a splice junction after their termination codon. We developed a genome-wide CRISPR flow cytometry screen to identify regulators of mRNAs with premature termination codons in K562 cells. This screen recovered essentially all core NMD factors and suggested a role for EJC factors in degradation of PTCs without downstream splicing. Among the strongest hits were the translational repressors GIGYF2 and EIF4E2. GIGYF2 and EIF4E2 mediate translational repression but not mRNA decay of a subset of NMD targets and interact with NMD factors genetically and physically. Our results suggest a model wherein recognition of a stop codon as premature can lead to its translational repression through GIGYF2 and EIF4E2.

携带提前终止密码子（premature translation termination codons, PTCs）的信使RNA（mRNA）经翻译后会产生截短型蛋白质，这类蛋白可能存在潜在有害效应。无义介导的mRNA降解（nonsense-mediated mRNA decay, NMD）可阻止此类情况发生。NMD的触发条件为核糖体在被外显子连接复合物（exon-junction complex, EJC）标记的剪接位点上游终止翻译，但NMD也可作用于诸多终止密码子下游不存在剪接接头的mRNA。我们开发了一种全基因组CRISPR流式细胞术筛选方法，用于鉴定K562细胞中调控携带提前终止密码子的mRNA的因子。该筛选几乎鉴定出了所有核心NMD因子，并提示EJC因子可在下游剪接缺失的情况下介导携带PTC的mRNA的降解。筛选得到的最显著命中因子包括翻译抑制因子GIGYF2与EIF4E2。GIGYF2与EIF4E2可介导部分NMD靶标的翻译抑制，但不影响其mRNA降解，且在遗传与物理层面均可与NMD因子发生相互作用。我们的研究结果提出了如下模型：将终止密码子识别为提前终止密码子的过程，可通过GIGYF2和EIF4E2引发其翻译抑制。