Assessment of selective mRNA translation in mammalian cells by polysome profiling.
Assessment of selective mRNA translation in mammalian cells by polysome profiling.
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DOI：
10.3791/52295
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发表时间：
2014-10-28
期刊：
影响因子：
--
通讯作者：
Holcik M
中科院分区：
文献类型：
--
作者：
Faye MD;Graber TE;Holcik M
蛋白质合成的调节是细胞对应激反应的关键控制点。特别是，谨慎的RNA调控元件被证明允许选择性地翻译特定的mRNAs，这些mRNAs通常编码特定应激反应所需的蛋白质。鉴定这些mRNAs，以及表征负责选择性翻译的调控机制，一段时间以来一直是分子生物学的前沿。在这些研究中，多聚体图谱是一种基石方法。多聚体图谱的目标是通过将翻译的核糖体主动固定在不同的转录本上来捕捉mRNA的翻译，并通过蔗糖梯度超速离心法分离得到的多聚核糖体，从而区分高翻译和低翻译的转录本。然后，可以用传统的生化和分子生物学方法进一步表征这些特征。重要的是，将多聚体图谱与高通量基因组方法相结合，可以对翻译调控进行大规模分析。
Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.
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