Mitochondrial Ribosome (Mitoribosome) Profiling for Monitoring Mitochondrial Translation In Vivo.
Mitochondrial Ribosome (Mitoribosome) Profiling for Monitoring Mitochondrial Translation In Vivo.
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DOI：
10.1002/cpmb.41
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发表时间：
2017-07-05
影响因子：
--
通讯作者：
Churchman, L Stirling
中科院分区：
文献类型：
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作者：
Couvillion, Mary T;Churchman, L Stirling
线粒体中的翻译受到与细胞质和细菌中不同的机制的调节，但研究它的精确方法已经落后。该单元描述了一种方法，线粒体核糖体（mitoribosome）的分析，以定量监测线粒体翻译与高时空分辨率的酿酒酵母。从全细胞裂解物中免疫沉淀线粒体，并分离受保护的mRNA片段。然后将这些片段转化为测序文库或通过北方印迹杂交分析，以揭示线粒体转录组中线粒体核糖体的分布。由于需要有关RNA丰度的信息来解决RNA效应的翻译，我们还提出了一种可以并行进行的RNA测序方法。准确捕捉生物相关的线粒体位置分布取决于几个关键参数进行了讨论。线粒体分析的应用可以揭示线粒体翻译控制的机制，这是以前不可能发现的。John Wiley & Sons，Inc.
Translation in the mitochondria is regulated by mechanisms distinct from those acting in the cytosol and in bacteria, yet precise methods for investigating it have lagged behind. This unit describes an approach, mitochondrial ribosome (mitoribosome) profiling, to quantitatively monitor mitochondrial translation with high temporal and spatial resolution in Saccharomyces cerevisiae. Mitoribosomes are immunoprecipitated from whole-cell lysate and the protected mRNA fragments are isolated. These fragments are then converted to sequencing libraries or analyzed by northern blot hybridization to reveal the distribution of mitoribosomes across the mitochondrial transcriptome. As information about RNA abundance is required to resolve translational from RNA effects, we also present an RNA sequencing approach that can be performed in parallel. Accurately capturing the biologically relevant distribution of mitoribosome positions depends on several critical parameters that are discussed. Application of mitoribosome profiling can reveal mechanisms of mitochondrial translational control that were not previously possible to uncover. © 2017 by John Wiley & Sons, Inc.