Ribosomal protein L6 (RPL6) is recruited to DNA damage sites in a poly(ADP-ribose) polymerase-dependent manner and regulates the DNA damage response
Ribosomal protein L6 (RPL6) is recruited to DNA damage sites in a poly(ADP-ribose) polymerase-dependent manner and regulates the DNA damage response
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核糖体蛋白 L6 (RPL6) 以聚 (ADP-核糖) 聚合酶依赖性方式募集到 DNA 损伤位点并调节 DNA 损伤反应
DOI：
10.1074/jbc.ra118.007009
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发表时间：
2019-02-22
影响因子：
4.8
通讯作者：
Zheng, Xiaofeng
中科院分区：
文献类型：
--
作者：
Yang, Chuanzhen;Zang, Weicheng;Zheng, Xiaofeng
核糖体蛋白是核糖体生物合成的基石。除了已知的参与核糖体组装外，核糖体蛋白质的核糖体非依赖性功能在很大程度上是未知的。在这里，使用免疫沉淀，亚细胞分级分离，组氨酸-泛素下拉，免疫荧光显微镜分析，沿着与基于siRNA的敲低方法，我们证明，核糖体蛋白L 6（RPL 6）直接与组蛋白H2 A相互作用，并参与DNA损伤反应（DDR）。我们发现，在DNA损伤的反应中，RPL 6以聚（ADP-核糖）聚合酶（PARP）依赖的方式被募集到DNA损伤位点，促进其与H2 A的相互作用。我们还观察到，RPL 6耗竭减弱了DNA损伤检查点1（MDC 1）和H2 A组蛋白家族成员X之间的相互作用，磷酸化（γ H2 AX），损害了MDC 1在DNA损伤位点的积累，并减少了环指蛋白168（RNF 168）和H2 A Lys-15泛素化（H2 AK 15 ub）的募集。这些RPL 6耗竭诱导的事件随后抑制了以下下游修复蛋白的募集：肿瘤蛋白P53结合蛋白1（TP 53 BP 1）和BRCA 1，DNA修复相关蛋白（BRCA 1）。此外，RPL 6敲低导致DNA损伤诱导的G（2）-M检查点、DNA损伤修复和细胞存活的缺陷。总之，我们的研究将RPL 6确定为参与DDR的关键调节因素。这些发现扩展了我们对核糖体蛋白在细胞生理学中的核糖体外功能的认识，加深了我们对DDR调控的分子机制的理解。
Ribosomal proteins are the building blocks of ribosome biogenesis. Beyond their known participation in ribosome assembly, the ribosome-independent functions of ribosomal proteins are largely unknown. Here, using immunoprecipitation, subcellular fractionation, His-ubiquitin pulldown, and immunofluorescence microscopy assays, along with siRNA-based knockdown approaches, we demonstrate that ribosomal protein L6 (RPL6) directly interacts with histone H2A and is involved in the DNA damage response (DDR). We found that in response to DNA damage, RPL6 is recruited to DNA damage sites in a poly(ADP-ribose) polymerase (PARP)-dependent manner, promoting its interaction with H2A. We also observed that RPL6 depletion attenuates the interaction between mediator of DNA damage checkpoint 1 (MDC1) and H2A histone family member X, phosphorylated (gamma H2AX), impairs the accumulation of MDC1 at DNA damage sites, and reduces both the recruitment of ring finger protein 168 (RNF168) and H2A Lys-15 ubiquitination (H2AK15ub). These RPL6 depletion-induced events subsequently inhibited the recruitment of the following downstream repair proteins: tumor protein P53-binding protein 1 (TP53BP1) and BRCA1, DNA repair-associated (BRCA1). Moreover, the RPL6 knockdown resulted in defects in the DNA damage-induced G(2)-M checkpoint, DNA damage repair, and cell survival. In conclusion, our study identifies RPL6 as a critical regulatory factor involved in the DDR. These findings expand our knowledge of the extraribosomal functions of ribosomal proteins in cell physiology and deepen our understanding of the molecular mechanisms underlying DDR regulation.