RNA-ID, a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay
RNA-ID, a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay
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DOI：
10.1261/rna.035907.112
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发表时间：
2012-12-01
期刊：
影响因子：
4.5
通讯作者：
Grayhack, Elizabeth J.
中科院分区：
文献类型：
--
作者：
Dean, Kimberly M.;Grayhack, Elizabeth J.
我们已经开发了一种强大而灵敏的方法，称为RNA-ID，使用携带报告基因的酵母细胞的荧光激活细胞分选（FACS）来筛选RNA中的顺式调节序列，其中超级折叠绿色荧光蛋白（GFP）和酵母密码子优化的mCherry红色荧光蛋白（RFP）的表达由双向GAL 1，10启动子驱动。该方法概括了先前报道的通过增加CGA密码子对的数量介导的翻译的渐进抑制，以及通过引入具有与CGA密码子完全碱基配对的反密码子的tRNA来恢复表达。该方法还再现了巴龙霉素和上下文对终止密码子通读的影响。该方法的五个关键特征有助于其作为调节序列选择的有效性：该系统表现出大于250倍的动态范围，对已知抑制序列的定量和剂量依赖性响应，允许不同群体几乎完全物理分离的精确分辨率，以及用相同报告基因转化的不同细胞之间的可再现信号，所有这些都与涉及不依赖连接克隆的简单方法相结合，以创建大型库。此外，我们提供的证据表明，有一个9-nt的库内的序列，导致减少GFP荧光，这表明有新的顺式调控序列被发现，即使在这个短的序列空间。该方法广泛适用于RNA介导和密码子介导的表达效应的研究。
We have developed a robust and sensitive method, called RNA-ID, to screen for cis-regulatory sequences in RNA using fluorescence-activated cell sorting (FACS) of yeast cells bearing a reporter in which expression of both superfolder green fluorescent protein (GFP) and yeast codon-optimized mCherry red fluorescent protein (RFP) is driven by the bidirectional GAL1,10 promoter. This method recapitulates previously reported progressive inhibition of translation mediated by increasing numbers of CGA codon pairs, and restoration of expression by introduction of a tRNA with an anticodon that base pairs exactly with the CGA codon. This method also reproduces effects of paromomycin and context on stop codon read-through. Five key features of this method contribute to its effectiveness as a selection for regulatory sequences: The system exhibits greater than a 250-fold dynamic range, a quantitative and dose-dependent response to known inhibitory sequences, exquisite resolution that allows nearly complete physical separation of distinct populations, and a reproducible signal between different cells transformed with the identical reporter, all of which are coupled with simple methods involving ligation-independent cloning, to create large libraries. Moreover, we provide evidence that there are sequences within a 9-nt library that cause reduced GFP fluorescence, suggesting that there are novel cis-regulatory sequences to be found even in this short sequence space. This method is widely applicable to the study of both RNA-mediated and codon-mediated effects on expression.