Elp1 通过翻译调控促进 RAD51 介导的同源重组修复。
Elp1 facilitates RAD51-mediated homologous recombination repair via translational regulation.
机构信息
Department of Biochemical Science and Technology, National Taiwan University, No.1, Sec.4, Roosevelt Rd., Taipei, 10617, Taiwan.
Institute of Biochemical Sciences, National Taiwan University, Taipei, 10617, Taiwan.
出版信息
J Biomed Sci. 2021 Nov 24;28(1):81. doi: 10.1186/s12929-021-00773-z.
BACKGROUND
RAD51-dependent homologous recombination (HR) is one of the most important pathways for repairing DNA double-strand breaks (DSBs), and its regulation is crucial to maintain genome integrity. Elp1 gene encodes IKAP/ELP1, a core subunit of the Elongator complex, which has been implicated in translational regulation. However, how ELP1 contributes to genome maintenance is unclear.
METHODS
To investigate the function of Elp1, Elp1-deficient mouse embryonic fibroblasts (MEFs) were generated. Metaphase chromosome spreading, immunofluorescence, and comet assays were used to access chromosome abnormalities and DSB formation. Functional roles of Elp1 in MEFs were evaluated by cell viability, colony forming capacity, and apoptosis assays. HR-dependent DNA repair was assessed by reporter assay, immunofluorescence, and western blot. Polysome profiling was used to evaluate translational efficiency. Differentially expressed proteins and signaling pathways were identified using a label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach.
RESULTS
Here, we report that Elp1 depletion enhanced genomic instability, manifested as chromosome breakage and genotoxic stress-induced genomic DNA fragmentation upon ionizing radiation (IR) exposure. Elp1-deficient cells were hypersensitive to DNA damage and exhibited impaired cell proliferation and defective HR repair. Moreover, Elp1 depletion reduced the formation of IR-induced RAD51 foci and decreased RAD51 protein levels. Polysome profiling analysis revealed that ELP1 regulated RAD51 expression by promoting its translation in response to DNA damage. Notably, the requirement for ELP1 in DSB repair could be partially rescued in Elp1-deficient cells by reintroducing RAD51, suggesting that Elp1-mediated HR-directed repair of DSBs is RAD51-dependent. Finally, using proteome analyses, we identified several proteins involved in cancer pathways and DNA damage responses as being differentially expressed upon Elp1 depletion.
CONCLUSIONS
Our study uncovered a molecular mechanism underlying Elp1-mediated regulation of HR activity and provides a novel link between translational regulation and genome stability.
背景
RAD51 依赖性同源重组（HR）是修复 DNA 双链断裂（DSB）的最重要途径之一，其调控对于维持基因组完整性至关重要。Elp1 基因编码 IKAP/ELP1，它是伸长因子复合物的核心亚基，已被牵涉到翻译调控中。然而，ELP1 如何促进基因组的维持尚不清楚。
方法
为了研究 Elp1 的功能，生成了 Elp1 缺陷型小鼠胚胎成纤维细胞（MEFs）。使用中期染色体铺展、免疫荧光和彗星试验来评估染色体异常和 DSB 的形成。通过细胞活力、集落形成能力和凋亡试验评估 Elp1 在 MEFs 中的功能作用。通过报告试验、免疫荧光和 Western blot 评估 HR 依赖性 DNA 修复。使用多核糖体谱分析来评估翻译效率。使用无标记液相色谱-串联质谱（LC-MS/MS）蛋白质组学方法鉴定差异表达蛋白和信号通路。
结果
在此，我们报告 Elp1 耗竭增强了基因组不稳定性，表现为染色体断裂和电离辐射（IR）暴露后遗传毒性应激诱导的基因组 DNA 片段化。Elp1 缺陷型细胞对 DNA 损伤敏感，并表现出增殖受损和 HR 修复缺陷。此外，Elp1 耗竭减少了 IR 诱导的 RAD51 焦点的形成，并降低了 RAD51 蛋白水平。多核糖体谱分析表明，ELP1 通过促进 RAD51 的翻译来调节 RAD51 表达，以响应 DNA 损伤。值得注意的是，在 Elp1 缺陷型细胞中，通过重新引入 RAD51 部分挽救了 ELP1 在 DSB 修复中的作用，这表明 Elp1 介导的 DSB 的 HR 定向修复是 RAD51 依赖性的。最后，通过蛋白质组分析，我们鉴定出几种涉及癌症途径和 DNA 损伤反应的蛋白在 Elp1 耗竭时差异表达。
结论
本研究揭示了 Elp1 介导的 HR 活性调节的分子机制，并为翻译调控与基因组稳定性之间提供了新的联系。
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