Analysis of translation using polysome profiling.
Analysis of translation using polysome profiling.
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DOI：
10.1093/nar/gkw907
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发表时间：
2017-02-17
影响因子：
14.9
通讯作者：
Morales J
中科院分区：
文献类型：
--
作者：
Chassé H;Boulben S;Costache V;Cormier P;Morales J
在过去的十年中，人们对许多生物体中基因表达翻译调控的作用越来越感兴趣。多核糖体分析已被开发用于推断特定 mRNA 种类的翻译状态或分析翻译组，即细胞中活跃翻译的 mRNA 子集。多核糖体分析特别适合基因组数据有限的新兴模式生物。在本文中，我们描述了海胆多核糖体纯化的优化方案，并重点介绍了多核糖体纯化中涉及的关键步骤。我们应用该协议来获得受精后 mRNA 翻译调控的实验结果。我们的协议对于整合任何生物模型中基因调控网络中翻译调控作用的研究应该是有用的。此外，我们还演示了如何对多核糖体梯度组分进行高通量处理，以同时筛选多种生物条件和大规模制备用于下一代测序的样品。
During the past decade, there has been growing interest in the role of translational regulation of gene expression in many organisms. Polysome profiling has been developed to infer the translational status of a specific mRNA species or to analyze the translatome, i.e. the subset of mRNAs actively translated in a cell. Polysome profiling is especially suitable for emergent model organisms for which genomic data are limited. In this paper, we describe an optimized protocol for the purification of sea urchin polysomes and highlight the critical steps involved in polysome purification. We applied this protocol to obtain experimental results on translational regulation of mRNAs following fertilization. Our protocol should prove useful for integrating the study of the role of translational regulation in gene regulatory networks in any biological model. In addition, we demonstrate how to carry out high-throughput processing of polysome gradient fractions, for the simultaneous screening of multiple biological conditions and large-scale preparation of samples for next-generation sequencing.