EC-tagging allows cell type-specific RNA analysis.
EC-tagging allows cell type-specific RNA analysis.
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DOI：
10.1093/nar/gkx551
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发表时间：
2017-09-06
影响因子：
14.9
通讯作者：
Cleary MD
中科院分区：
文献类型：
--
作者：
Hida N;Aboukilila MY;Burow DA;Paul R;Greenberg MM;Fazio M;Beasley S;Spitale RC;Cleary MD
细胞类型特异性RNA的纯化仍然是一个重大挑战。一种解决方案涉及靶RNA的生物合成标记。通过在表达转基因尿嘧啶磷酸核糖基转移酶（UPRT）的细胞中掺入4-硫氧嘧啶（TU）的RNA标记（一种称为TU标记的方法）已用于多个系统中，但由于TU掺入的内源性途径而具有有限的特异性。在这里，我们描述了一种替代方法，需要两种酶的活性：胞嘧啶脱氨酶（CD）和UPRT。我们发现，这些酶的连续活性将5-乙炔基胞嘧啶（EC）转化为5-乙炔基尿苷一磷酸，随后将其掺入新生RNA中。乙炔基允许有效地检测和纯化标记的RNA。我们表明，“EC标记”发生在组织培养细胞和果蝇工程表达CD和UPRT。另外的控制可以通过分裂CD方法来实现，其中功能性CD从独立表达的片段重构。我们通过从完整的果蝇幼虫获得细胞类型特异性基因表达数据，包括从一小群中枢脑神经元的转录组测量，证明了EC标记的灵敏度和特异性。EC标记提供了几个优势，现有的技术，并应广泛用于调查的作用，差异RNA表达在细胞的身份，生理和病理。
Purification of cell type-specific RNAs remains a significant challenge. One solution involves biosynthetic tagging of target RNAs. RNA tagging via incorporation of 4-thiouracil (TU) in cells expressing transgenic uracil phosphoribosyltransferase (UPRT), a method known as TU-tagging, has been used in multiple systems but can have limited specificity due to endogenous pathways of TU incorporation. Here, we describe an alternative method that requires the activity of two enzymes: cytosine deaminase (CD) and UPRT. We found that the sequential activity of these enzymes converts 5-ethynylcytosine (EC) to 5-ethynyluridine monophosphate that is subsequently incorporated into nascent RNAs. The ethynyl group allows efficient detection and purification of tagged RNAs. We show that ‘EC-tagging’ occurs in tissue culture cells and Drosophila engineered to express CD and UPRT. Additional control can be achieved through a split-CD approach in which functional CD is reconstituted from independently expressed fragments. We demonstrate the sensitivity and specificity of EC-tagging by obtaining cell type-specific gene expression data from intact Drosophila larvae, including transcriptome measurements from a small population of central brain neurons. EC-tagging provides several advantages over existing techniques and should be broadly useful for investigating the role of differential RNA expression in cell identity, physiology and pathology.
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