Juntawong Piyada, Hummel Maureen, Bazin Jeremie, Bailey-Serres Julia
Center for Plant Cell Biology and Department of Botany and Plant Sciences, University of California, Riverside, CA, 92521, USA.
Methods Mol Biol. 2015;1284:139-73. doi: 10.1007/978-1-4939-2444-8_7.
Translational regulation is important for plant growth, metabolism, and acclimation to environmental challenges. Ribosome profiling involves the nuclease digestion of mRNAs associated with ribosomes and mapping of the generated ribosome-protected footprints to transcripts. This is useful for investigation of translational regulation. Here we present a detailed method to generate, purify, and high-throughput-sequence ribosome footprints from Arabidopsis thaliana using two different isolation methods, namely, conventional differential centrifugation and the translating ribosome affinity purification (TRAP) technology. These methodologies provide researchers with an opportunity to quantitatively assess with high-resolution the translational activity of individual mRNAs by determination of the position and number of ribosomes in the corresponding mRNA. The results can provide insights into the translation of upstream open reading frames, alternatively spliced transcripts, short open reading frames, and other aspects of translation.
翻译调控对植物生长、代谢以及适应环境挑战至关重要。核糖体谱分析涉及对与核糖体相关的mRNA进行核酸酶消化，并将产生的核糖体保护片段定位到转录本上。这对于研究翻译调控很有用。在这里，我们介绍一种详细的方法，使用两种不同的分离方法，即传统的差速离心法和翻译核糖体亲和纯化（TRAP）技术，从拟南芥中生成、纯化核糖体足迹并进行高通量测序。这些方法为研究人员提供了一个机会，通过确定相应mRNA中核糖体的位置和数量，以高分辨率定量评估单个mRNA的翻译活性。结果可以为上游开放阅读框的翻译、可变剪接转录本、短开放阅读框以及翻译的其他方面提供见解。
