RNA-protein analysis using a conditional CRISPR nuclease
RNA-protein analysis using a conditional CRISPR nuclease
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DOI：
10.1073/pnas.1302807110
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发表时间：
2013-04-02
影响因子：
11.1
通讯作者：
Doudna, Jennifer A.
中科院分区：
文献类型：
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作者：
Lee, Ho Young;Haurwitz, Rachel E.;Doudna, Jennifer A.
RNA结合蛋白控制着所有细胞中转录组的命运和功能。在这里，我们介绍了使用工程化的簇状规则间隔短回文重复序列(CRISPR)内切核糖核酸酶高效而准确地分离RNA-蛋白质伴侣的技术。Csy4核酸酶的失活版本不可逆地与转录产物结合，这些转录产物在其5‘端带有16个核苷酸的发夹序列。一旦被Csy4固定在固体载体上，污染的蛋白质和其他分子可以通过广泛的洗涤来去除。在加入咪唑后，Csy4被激活以切割RNA，去除发夹标签并释放天然转录本与其特定结合的蛋白质伙伴。这种有条件的Csy4酶能够恢复特定的RNA结合伙伴，并将假阳性污染降至最低。我们使用这种方法，结合定量MS，来鉴定特定细胞类型的人类前microRNA结合蛋白。我们还表明，该技术适用于分析不同大小的转录，并且适合于适应高通量的发现格式。
RNA-binding proteins control the fate and function of the transcriptome in all cells. Here we present technology for isolating RNA-protein partners efficiently and accurately using an engineered clustered regularly interspaced short palindromic repeats (CRISPR) endoribonuclease. An inactive version of the Csy4 nuclease binds irreversibly to transcripts engineered with a 16-nt hairpin sequence at their 5' ends. Once immobilized by Csy4 on a solid support, contaminating proteins and other molecules can be removed by extensive washing. Upon addition of imidazole, Csy4 is activated to cleave the RNA, removing the hairpin tag and releasing the native transcript along with its specifically bound protein partners. This conditional Csy4 enzyme enables recovery of specific RNA-binding partners with minimal false-positive contamination. We use this method, coupled with quantitative MS, to identify cell type-specific human pre-microRNA-binding proteins. We also show that this technology is suitable for analyzing diverse size transcripts, and that it is suitable for adaptation to a high-throughput discovery format.