Visualization of Mitochondrial RNA Granules in Cultured Cells Using 5-Bromouridine Labeling.
Visualization of Mitochondrial RNA Granules in Cultured Cells Using 5-Bromouridine Labeling.
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使用 5-溴尿苷标记可视化培养细胞中的线粒体 RNA 颗粒。
DOI：
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发表时间：
2021
影响因子：
--
通讯作者：
J. Martinou
中科院分区：
文献类型：
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作者：
V. J. Xavier;J. Martinou
核苷类似物的掺入是研究DNA和RNA的各种功能的有用工具。这些类似物可以通过荧光或通过免疫标记直接检测，从而允许可视化、追踪或测量它们已经掺入其中的核酸分子。在这一章中，方法来衡量人类线粒体转录。从线粒体DNA（mtDNA）转录的新生RNA已被证明组装成大的核糖核蛋白复合物，形成离散的焦点。这些结构被称为线粒体RNA颗粒（MRG），可以通过掺入5-溴尿苷（BrU）在体外观察到，随后通过荧光免疫标记可视化。在这里，详细介绍了一种用于MRG检测的组合方案，包括BrU标记和可视化其真正的蛋白质组分之一，Fas激活的丝氨酸-苏氨酸激酶结构域2（FASTKD 2）。基于免疫检测，在各种实验条件下MRG的半衰期和动力学可以通过用过量的尿苷追踪BrU脉冲来进一步确定。
The incorporation of nucleoside analogs is a useful tool to study the various functions of DNA and RNA. These analogs can be detected directly by fluorescence or by immunolabeling, allowing to visualize, track, or measure the nucleic acid molecules in which they have been incorporated. In this chapter, methodologies to measure human mitochondrial transcription are described. The nascent RNA that is transcribed from mitochondrial DNA (mtDNA) has been shown to assemble into large ribonucleoprotein complexes that form discrete foci. These structures were called mitochondrial RNA granules (MRGs) and can be observed in vitro by the incorporation of a 5-Bromouridine (BrU), which is subsequently visualized by fluorescent immunolabeling. Here, a combined protocol for the MRGs detection is detailed, consisting of BrU labeling and visualization of one of their bona fide protein components, Fas-activated serine-threonine kinase domain 2 (FASTKD2). Based on immunodetection, the half-life and kinetics of the MRGs under various experimental conditions can further be determined by chasing the BrU pulse with an excess of Uridine.
影响因子：
8.8
作者：
Tu YT;Barrientos A
通讯作者：
Barrientos A