PDF(Pubmed)
Abstract：
We have previously developed a transcription-based bacterial three-hybrid (B3H) assay as a genetic approach to probe RNA-protein interactions inside of E. coli cells. This system offers a straightforward path to identify and assess the consequences of mutations in RBPs with molecular phenotypes of interest. One limiting factor in detecting RNA-protein interactions in the B3H assay is RNA misfolding arising from incorrect base-pair interactions with neighboring RNA sequences in a hybrid RNA. To support correct folding of hybrid bait RNAs, we have explored the use of a highly stable stem (\"GC clamp\") to isolate regions of a hybrid RNA as discrete folding units. In this work, we introduce new bait RNA constructs to 1) insulate the folding of individual components of the hybrid RNA with GC clamps and 2) express bait RNAs that do not encode their own intrinsic terminator. We find that short GC clamps (5 or 7 bp long) are more effective than a longer 13bp GC clamp in the B3H assay. These new constructs increase the number of Hfq-sRNA and -5\'UTR interactions that are detectable in the B3H system and improve the signal-to-noise ratio of many of these interactions. We therefore recommend the use of constructs containing short GC clamps for the expression of future B3H bait RNAs. With these new constructs, a broader range of RNA-protein interactions are detectable in the B3H assay, expanding the utility and impact of this genetic tool as a platform to search for and interrogate mechanisms of additional RNA-protein interactions.
摘要：
我们以前已经开发了一种基于转录的细菌三杂交（B3H）测定法，作为一种遗传方法来探测大肠杆菌细胞内部的RNA-蛋白质相互作用。该系统提供了一条直接的途径来鉴定和评估具有感兴趣的分子表型的RBP中突变的后果。在B3H测定中检测RNA-蛋白质相互作用的一个限制因素是由与杂合RNA中的相邻RNA序列的不正确碱基对相互作用引起的RNA错误折叠。为了支持杂交诱饵RNA的正确折叠，我们已经探索了使用高度稳定的茎（“GC钳”）来分离杂合RNA的区域作为离散的折叠单元。在这项工作中,我们引入了新的诱饵RNA构建体，以1）用GC夹钳隔离杂交RNA的各个成分的折叠，以及2）表达不编码其自身固有终止子的诱饵RNA。我们发现在B3H测定中,短GC夹钳(5或7bp长)比长13bpGC夹钳更有效。这些新构建体增加了在B3H系统中可检测到的Hfq-sRNA和-5'UTR相互作用的数量，并改善了许多这些相互作用的信噪比。因此，我们建议使用包含短GC夹钳的构建体来表达未来的B3H诱饵RNA。有了这些新结构，在B3H分析中可以检测到更广泛的RNA-蛋白质相互作用，扩大这种遗传工具的效用和影响，作为搜索和询问额外RNA-蛋白质相互作用机制的平台。
