Dynamic Behavior of the RNA Polymerase II and the Ubiquitin Proteasome System During the Neuronal DNA Damage Response to Ionizing Radiation
Dynamic Behavior of the RNA Polymerase II and the Ubiquitin Proteasome System During the Neuronal DNA Damage Response to Ionizing Radiation
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DOI：
10.1007/s12035-015-9565-8
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发表时间：
2016-12-01
影响因子：
5.1
通讯作者：
Lafarga, Miguel
中科院分区：
文献类型：
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作者：
Casafont, Inigo;Palanca, Ana;Lafarga, Miguel
神经元非常容易受到基因毒性物质的影响。为了在DNA损伤后恢复基因组的完整性，神经元触发DNA损伤反应(DDR)，这需要染色质修饰和DNA损伤部位的转录沉默。为了研究转录所有mRNA编码基因的活性RNA聚合酶II(POL II)的重组以及泛素-蛋白酶体系统(UPS)在神经元DDR中的参与，我们利用X射线(4GY)电离辐射(IR)对大鼠感觉神经节神经元进行了研究。在对照神经元中，Pol II集中在许多染色质微灶中，通过将5‘-氟尿嘧啶掺入新生RNA中而被鉴定为转录工厂。在IR治疗后，早在IR后30min就可观察到大量IR诱导灶(IRIF)，其数量在IR后3h、1d和3d逐渐减少，且对γ-H2AX和53BP1呈免疫反应。IRIF的形成与免疫荧光和Western blotting检测到的POL II水平降低有关。蛋白酶体抑制剂Bortezomib显著增加了对照和照射神经元中Pol II的水平，表明蛋白酶体通过清除DNA损伤部位停滞的Pol II复合体发挥蛋白分解作用，作为DNA修复的前奏。神经元IRIF募集泛素化的蛋白质，包括泛素化的组蛋白H_2A(Ub-H_2A)和催化蛋白酶体20S。UB-H2A与DNA损伤部位的转录沉默有关。另一方面，UPS在神经元DDR中的参与对于Pol II和DNA修复机制的其他蛋白酶体底物的泛素化及其随后的蛋白酶体介导的降解可能是必不可少的。
Neurons are highly vulnerable to genotoxic agents. To restore genome integrity upon DNA lesions, neurons trigger a DNA damage response (DDR) that requires chromatin modifications and transcriptional silencing at DNA damage sites. To study the reorganization of the active RNA polymerase II (Pol II), which transcribes all mRNA-encoding genes, and the participation of the ubiquitin-proteasome system (UPS) in the neuronal DDR, we have used rat sensory ganglion neurons exposed to X-rays (4 Gy) ionizing radiation (IR). In control neurons, Pol II appears concentrated in numerous chromatin microfoci identified as transcription factories by the incorporation of 5'-fluorouridine into nascent RNA. Upon IR treatment, numerous IR-induced foci (IRIF), which were immunoreactive for gamma H2AX and 53BP1, were observed as early as 30 min post-IR; their number progressively reduced at 3 h, 1 day, and 3 days post-IR. The formation of IRIF was associated with a decrease in Pol II levels by both immunofluorescence and Western blotting. Treatment with the proteasome inhibitor bortezomib strongly increased Pol II levels in both control and irradiated neurons, suggesting that proteasome plays a proteolytic role by clearing stalled Pol II complexes at DNA damage sites, as a prelude to DNA repair. Neuronal IRIF recruited ubiquitylated proteins, including ubiquitylated histone H2A (Ub-H2A), and the catalytic proteasome 20S. Ub-H2A has been associated with transcriptional silencing at DNA damage sites. On the other hand, the participation of UPS in neuronal DDR may be essential for the ubiquitylation of Pol II and other proteasome substrates of the DNA repair machinery and their subsequent proteasome-mediated degradation.