Nuclear organization and dynamics of transcription sites in rat sensory ganglia neurons detected by incorporation of 5′-fluorouridine into nascent RNA
Nuclear organization and dynamics of transcription sites in rat sensory ganglia neurons detected by incorporation of 5′-fluorouridine into nascent RNA
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DOI：
10.1016/j.neuroscience.2006.02.030
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发表时间：
2006-01-01
期刊：
影响因子：
3.3
通讯作者：
Berciano, M. T.
中科院分区：
文献类型：
--
作者：
Casafont, I.;Navascues, J.;Berciano, M. T.
在这项研究中，我们使用了转录检测与5 '-氟尿嘧啶掺入新生RNA分析核组织和转录位点的动力学在大鼠三叉神经节神经元。腹腔注射给药的5 '-FU成功地掺入到含有三叉神经元活性转录基因的核域中。用免疫细胞化学方法在光镜和电镜水平检测5 '-氟尿苷RNA标记。在核仁中检测到5 '-氟尿苷掺入位点，特别是在致密的纤维状组分上，并且在遍布常染色质区域的许多转录灶中检测到5'-氟尿苷掺入位点，而没有优先定位在核周边或核内部。联合收割机结合5 '-氟尿苷掺入与核区室分子标记的双标记实验表明，在Cajal体和剪接因子的核斑点中缺乏转录位点。类似地，在充分表征的染色质沉默结构域（端粒异染色质）中未检测到5 '-氟尿苷标记。通过腹膜内给予转录抑制剂放线菌素D，验证了三叉神经节神经元中连续转录测定的特异性和灵敏度。放线菌素D处理后RNA合成的急剧减少与两个重要的细胞事件相关，异染色质沉默和DNA损伤/修复核灶的形成，分别通过三甲基化组蛋白H4和磷酸化H2 AX的表达来证明。动物模型中的5 '-氟尿苷掺入提供了一种有用的工具，用于研究正常生理学和实验病理学系统中哺乳动物神经元中基因表达的组织，（c）2006 IBRO。由爱思唯尔有限公司出版。保留所有权利。
In this study we have used the transcription assay with 5'-fluorouridine incorporation into nascent RNA to analyze the nuclear organization and dynamics of transcription sites in rat trigeminal ganglia neurons. The 5'-FU administrated by i.p. injection was successfully incorporated into nuclear domains containing actively transcribing genes of trigeminal neurons. 5'-Fluorouridine RNA-labeling was detected with immunocytochemistry at light and electron microscopy levels. The 5'-fluorouridine incorporation sites were detected in the nucleolus, particularly on the dense fibrillar component, and in numerous transcription foci spread throughout the euchromatin regions, without preferential positioning at the nuclear periphery or in the nuclear interior. Double labeling experiments to combine 5'-fluorouridine incorporation with molecular markers of nuclear compartments showed the absence of transcription sites in Cajal bodies and nuclear speckles of splicing factors. Similarly, no 5'-fluorouridine labeling was detected in well-characterized chromatin silencing domain, the telomeric heterochromatin. The specificity and sensitivity of the run-on transcription assay in trigeminal ganglia neurons was verified by the i.p. administration of the transcription inhibitor actinomycin D. The dramatic reduction in RNA synthesis upon actinomycin D treatment was associated with two important cellular events, heterochromatin silencing and formation of DNA damage/repair nuclear foci, demonstrated by the expression of tri-methylated histone H4 and phosphorylated H2AX, respectively. 5'-Fluorouridine incorporation in animal models provides a useful tool to investigate the organization of gene expression in mammalian neurons in both normal physiology and experimental pathology systems, (c) 2006 IBRO. Published by Elsevier Ltd. All rights reserved.