Translational inhibition by Schlafen proteins during the DNA damage response

最近，人类Schlafen 11(SLFN11)-我们已经证明它可以抑制HIV蛋白的表达，因为 研究发现，病毒独特的密码子使用偏向决定了细胞在暴露于DNA损伤后的命运 代理(DDA)。缺乏SLFN11的细胞对DDAs耐药，但对其他化疗药物不耐药。AS DDA是最大的抗癌药物，对它们的耐药性影响着大量的患者和 因此，揭示SLFN11‘S分子对DDAs疗效的贡献，并将其恢复到W/O细胞中是至关重要的。 SLFN11.到目前为止，SLFN11缺失导致对DDA产生抗性的事件仍未得到答复。 我们现在证明SLFN11抑制ATR翻译以响应DDAS来增强细胞杀伤。这 识别抑制翻译是由于在ATR中突出使用了特定的亮氨酸密码子。SLFN11抑制 当Leu(经常)通过TTA或CTT编码时翻译，但当使用其他密码子时不翻译。我们 展示了DDA诱导、SLFN11介导的对包括tRNAs Leu-TAA在内的不同tRNA亚群的切割 和Leu-AAG。SLFn11基因缺陷细胞的DDA敏感性可通过取消ATR的表达来恢复；2) 通过抑制ATR激酶活性；或3)通过使用Gapmer，这是我们适应的一种新技术 选择性靶向tRNA Leu-TAA进行降解。我们注意到了一种新的密码子特异性调控机制 SLFN11在DNA损伤反应中的翻译存在，并提供了第一个证据，表明 不同的tRNA允许依赖这些tRNA靶向特定的蛋白质。我们提供概念验证， 通过Gapmer靶向tRNAs是操纵细胞生存或病毒复制等行为的有效方法。 我们的首要目标是提高我们对SLFN11的功能和调控的了解 在细胞和分子水平上的DNA损伤反应。目的1重点分析SLFN11本身， 探索其功能域和调控机制。我们已经确定了几个抑制磷酸化的位点 SLFn11意味着SLFN11的激活需要去磷酸化，并表明PP1Cγ是 在DNA损伤反应中激活磷酸酶。这些发现需要得到验证和扩展 在其他设置中启用(其他小区类型和DDA？检查可能的额外(去)磷酸化 网站？确定可能的辅助因素？) 目标2中概述的实验针对tRNA裂解的作用(识别裂解位点(S)；测试 TRNAs转录后修饰的潜在要求；抗切割tRNA Leu-TAA 突变体使细胞对DDA产生抗药性，这样的“突变体”在自然界中是否存在？可能的生物学功能 TRNA衍生的核酸片段？)。 拟议研究的成功完成将支持SLFN11缺陷的癌细胞 可以通过靶向ATR或不同的tRNA以及特定类型的抑制来对DDA治疗(重新)敏感 II tRNAs可能为克服对DDAs的抗性提供一种新的策略。最后，在我们的艾滋病毒研究中，我们发现 SLFN11在逆转录/长病毒感染过程中抑制病毒蛋白的翻译，但不抑制其他病毒的翻译。我们 现在认为造成这种现象的原因是逆转录病毒在整合过程中导致DNA损伤， 从而可能激活SLFN11。相比之下，例如流感(尽管密码子偏向与艾滋病毒相似)却不是 受SLFN11抑制。我们现在假设SLFN11实际上可能抑制流感或其他“有偏见的” 如果SLFN11是通过药理手段独立激活的，则病毒。

Recently, human Schlafen 11 (SLFN11) - which we had shown to inhibit HIV protein expression due to the distinct codon-usage bias of the virus – was found to determine cell fate after exposure to DNA-damaging agents (DDAs). Cells lacking SLFN11 are resistant to DDAs, but not to other chemotherapeutic drugs. As DDAs are the largest group of cancer drugs, resistance against them impacts a large patient population and thus it is vital to unravel Slfn11's molecular contribution to the efficacy of DDAs, and to restore it in cells w/o Slfn11. So far, the events by which loss of SLFN11 causes resistance to DDAs remained unanswered. We now show that SLFN11 inhibits ATR translation in response to DDAs to enhance cell killing. This discerning inhibition translation is due to the prominent use of specific Leu codons in ATR. SLFN11 inhibits translation when Leu is (frequently) encoded via TTA or CTT, but not when other codons are employed. We demonstrate DDA-induced, SLFN11-mediated cleavage of a distinct tRNA subset including tRNAs Leu-TAA and Leu-AAG. DDA sensitivity in Slfn11-deficient cells can be restored 1) by abrogation of ATR expression; 2) through inhibition of ATR kinase activity; or 3) through the use of Gapmers, a novel technology we adapted to selectively target tRNA Leu-TAA for degradation. We note a novel mechanism of codon-specific regulation of translation by SLFN11 in the DNA damage response exists and provides the first evidence that modulation of a distinct tRNA allows for targeting specific proteins relying on those tRNAs. We provide proof-of-concept that targeting tRNAs by Gapmers is a valid approach to manipulate actions such as cell survival or viral replication. Our overarching goal is to improve our understanding of the function and regulation of Slfn11 during the DNA damage response on a cellular and molecular level. Aim 1 focuses on the analysis of Slfn11 itself, exploring its functional domains and regulation. We already identified several inhibitory phosphorylation sites in Slfn11 implying that dephosphorylation is required for SLFN11 activation, and show that PP1Cγ is the activating phosphatase during the DNA damage response. These findings need to be verified and expanded upon in additional settings (Other cell types and DDAs? Check for possible additional (de)phosphorylation sites? Identify likely cofactors?) The experiments outlined in Aim 2 target the role of the tRNA cleavage (identify cleavage site(s); test potential requirement for post-transcriptional modifications of tRNAs; do cleavage-resistant tRNA Leu-TAA mutants render cells DDA-resistant, and do such “mutants” exist in nature? Possible biological function for the tRNA-derived nucleic acid fragments?). Successful completion of the proposed studies will support the notion that SLFN11-deficient cancer cells can be (re)sensitized to DDA therapy by targeting ATR or distinct tRNAs, and that suppression of specific type II tRNAs might offer a new strategy to overcome resistance to DDAs. Finally, in our HIV studies we found that SLFN11 inhibited the translation of viral proteins during retro/lenti-viral infections, but not other viruses. We now think that the reason for this phenomenon is that retro-viruses cause DNA damage during integration, thereby likely activating SLFN11. In contrast, e.g. Influenza (despite similarity in codon bias to HIV) was not inhibited by SLFN11. We hypothesize now that SLFN11 might actually inhibit Influenza or other “biased” viruses if SLFN11 is independently activated by pharmacological means.