Nguyen Linh D, LeBlanc Hannah, Berry Katherine E
Program in Biochemistry, Mount Holyoke College, South Hadley, MA, 01075, USA.
Department of Chemistry, Mount Holyoke College, South Hadley, MA, 01075, USA.
bioRxiv. 2024 Jul 23:2024.07.23.604302. doi: 10.1101/2024.07.23.604302.
We have previously developed a transcription-based bacterial three-hybrid (B3H) assay as a genetic approach to probe RNA-protein interactions inside of 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.
我们之前开发了一种基于转录的细菌三杂交（B3H）检测方法，作为一种在细胞内探测RNA-蛋白质相互作用的遗传学方法。该系统为鉴定和评估具有感兴趣分子表型的RNA结合蛋白（RBP）突变的后果提供了一条直接途径。B3H检测中检测RNA-蛋白质相互作用的一个限制因素是杂交RNA中由于与相邻RNA序列的不正确碱基对相互作用而产生的RNA错误折叠。为了支持杂交诱饵RNA的正确折叠，我们探索了使用高度稳定的茎（“GC夹”）将杂交RNA区域分离为离散的折叠单元。在这项工作中，我们引入了新的诱饵RNA构建体，以1）用GC夹隔离杂交RNA各个组分的折叠，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检测中，短GC夹（5或7个碱基对长）比更长的13个碱基对的GC夹更有效。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，并改善了其中许多相互作用的信噪比。因此，我们建议使用含有短GC夹的构建体来表达未来的B3H诱饵RNA。有了这些新构建体，在B3H检测中可以检测到更广泛的RNA-蛋白质相互作用，扩大了这种遗传工具作为寻找和探究其他RNA-蛋白质相互作用机制平台的效用和影响。
