Active Ribosome Profiling with RiboLace
Active Ribosome Profiling with RiboLace
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DOI：
10.1016/j.celrep.2018.09.084
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发表时间：
2018-10-23
期刊：
影响因子：
8.8
通讯作者：
Viero, Gabriella
中科院分区：
文献类型：
--
作者：
Clamer, Massimiliano;Tebaldi, Toma;Viero, Gabriella
核糖体分析 (Ribo-seq) 基于对免受核糖体核酸酶消化的 RNA 片段进行大规模测序。由于其独特的能力提供有关沿着转录本流动的核糖体的位置信息，该方法可用于阐明翻译的机械方面。然而，当前的 Ribo-seq 方法缺乏区分受活跃翻译中的核糖体或非活性核糖体保护的片段的能力。为了克服这一可能的限制，我们开发了 RiboLace，一种基于原始含嘌呤霉素分子的方法，能够通过无抗体和无标签的下拉方法分离活性核糖体。 RiboLace 速度快，只需少量输入材料即可可靠工作，并且可以轻松快速地应用于体外和体内，从而以单核苷酸分辨率生成活性核糖体足迹的全局快照。
Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possible limitation, we developed RiboLace, a method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.