Velcrin-induced selective cleavage of tRNALeu(TAA) by SLFN12 causes cancer cell death [Ribo-Seq]

Velcrin compounds kill cancer cells expressing high levels of phosphodiesterase 3A (PDE3A) and Schlafen family member 12 (SLFN12) by inducing complex formation between these two proteins, but the mechanism of cancer cell killing by the PDE3A–SLFN12 complex is not fully understood. Here, we report that the physiological substrate of SLFN12 RNase is tRNALeu(TAA). SLFN12 selectively digests tRNALeu(TAA), and velcrin treatment promotes the cleavage of tRNALeu(TAA) by inducing PDE3A–SLFN12 complex formation in vitro. We found that distinct sequences in the variable loop and acceptor stem of tRNALeu(TAA) are required for substrate digestion. Velcrin treatment of sensitive cells results in downregulation of tRNALeu(TAA), ribosome pausing at Leu-TTA codons and global inhibition of protein synthesis. Velcrin-induced cleavage of tRNALeu(TAA) by SLFN12 and the concomitant global inhibition of protein synthesis thus define a new mechanism of apoptosis initiation. Overall design: Ribosome profiling of Hela cells treated with DMSO control or DNMDP in duplicate

Velcrin化合物可通过诱导磷酸二酯酶3A（PDE3A）与Schlafen家族成员12（SLFN12）形成复合物，从而杀伤高表达这两种蛋白的癌细胞，但PDE3A–SLFN12复合物介导的癌细胞杀伤机制尚未完全阐明。本研究证实，SLFN12核糖核酸酶（RNase）的生理底物为反密码子为TAA的亮氨酸转运RNA（tRNALeu(TAA)）。SLFN12可选择性降解tRNALeu(TAA)，而Velcrin处理可在体外通过诱导PDE3A–SLFN12复合物的形成，促进tRNALeu(TAA)的切割。研究发现，tRNALeu(TAA)的可变环与接受茎中的特定序列是其作为底物被降解的必要条件。对敏感细胞施加Velcrin处理后，可观察到tRNALeu(TAA)水平下调、核糖体在亮氨酸-TTA密码子处发生暂停，以及全局蛋白质合成受到抑制。综上，Velcrin通过诱导SLFN12切割tRNALeu(TAA)并伴随全局蛋白质合成抑制，这一过程定义了一种全新的细胞凋亡启动机制。实验设计概述：对经二甲基亚砜（DMSO）对照或DNMDP处理的海拉细胞开展核糖体谱分析，每组设置两个生物学重复。