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RNA-ID, a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay

机译:RNA-ID,一种使用超级文件夹GFP和基于荧光的测定法来鉴定顺式调控序列的高度灵敏且可靠的方法

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摘要

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.
机译:我们已经开发了一种健壮且灵敏的方法,称为RNA-ID,可使用带有报告基因的酵母细胞的荧光激活细胞分选(FACS)筛选RNA中的顺式调节序列,其中两种超文件夹绿色荧光蛋白(GFP)的表达酵母密码子优化的mCherry红色荧光蛋白(RFP)由双向GAL1,10启动子驱动。该方法概括了以前报道的通过抑制CGA密码子对数目的增加而介导的翻译的逐步抑制,以及通过引入带有与CGA密码子完全配对的反密码子的tRNA来恢复表达的方法。此方法还重现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为其选择调节序列的有效性做出了贡献:该系统展现出超过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,精湛的分离度,可实现几乎完全的物理分离不同的种群,以及用同一报告基因转化的不同细胞之间的可再现信号,所有这些都与涉及连接无关克隆的简单方法结合在一起,以创建大型文库。此外,我们提供的证据表明9-nt文库中存在导致GFP荧光降低的序列,这表明即使在此短序列空间中也存在新的顺式调控序列。该方法可广泛用于研究RNA介导的和密码子介导的表达影响。

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