Dutta Debapratim, Belashov Ivan A, Wedekind Joseph E
Department of Biochemistry & Biophysics and Center for RNA Biology , University of Rochester School of Medicine & Dentistry , Rochester , New York 14642 , United States.
Biochemistry. 2018 Aug 7;57(31):4620-4628. doi: 10.1021/acs.biochem.8b00316. Epub 2018 Jun 26.
Noncoding RNAs engage in numerous biological activities including gene regulation. To fully understand RNA function it is necessary to probe biologically relevant conformations in living cells. To address this challenge, we coupled RNA-mediated regulation of the green fluorescent protein (GFP)uv-reporter gene to icSHAPE (in cell Selective 2'-Hydroxyl Acylation analyzed by Primer Extension). Our transcript-specific approach provides sensitive, fluorescence-based readout of the regulatory-RNA status as a means to coordinate chemical modification experiments. We chose a plasmid-based reporter compatible with Escherichia coli to allow use of knockout strains that eliminate endogenous effector biosynthesis. The approach was piloted using the Lactobacillus rhamnosus ( Lrh) preQ-II riboswitch, which senses the pyrrolopyrimidine metabolite preQ. Using an E. coli Δ queF strain incapable of preQ anabolism, the Lrh riboswitch yielded nearly one log unit of GFPuv-gene repression resulting from exogenously added preQ. We then subjected cells in gene "on" and "off" states to icSHAPE. The resulting differential analysis indicated reduction in Lrh riboswitch flexibility in the P3 helix of the pseudoknot, which comprises the ribosome-binding site (RBS) paired with the anti-RBS. Such expression platform modulation was not observed by in vitro chemical probing and demonstrates that the crowded cellular environment does not preclude detection of compact and loose RNA-regulatory conformations. Here we describe the design, methods, interpretation, and caveats of Reporter Coupled (ReCo) icSHAPE. We also describe mapping of the differential ReCo-icSHAPE results onto the Lrh riboswitch-preQ cocrystal structure. The approach should be readily applicable to functional RNAs triggered by effectors or environmental variations.
非编码RNA参与包括基因调控在内的众多生物学活动。为了全面了解RNA功能，有必要在活细胞中探究生物学相关构象。为应对这一挑战，我们将RNA介导的绿色荧光蛋白（GFP）uv报告基因调控与icSHAPE（通过引物延伸分析细胞内选择性2'-羟基酰化）相结合。我们的转录本特异性方法提供了基于荧光的灵敏读数，用于反映调控RNA的状态，以此来协调化学修饰实验。我们选择了一种与大肠杆菌兼容的基于质粒的报告基因，以便能够使用消除内源性效应物生物合成的基因敲除菌株。该方法以鼠李糖乳杆菌（Lrh）前体Q-II核糖开关进行了试点，该核糖开关可感知吡咯并嘧啶代谢物前体Q。使用无法合成前体Q的大肠杆菌ΔqueF菌株，外源性添加的前体Q使Lrh核糖开关导致GFPuv基因的抑制接近一个对数单位。然后，我们对处于基因“开启”和“关闭”状态的细胞进行icSHAPE分析。所得的差异分析表明，假结的P3螺旋中Lrh核糖开关的灵活性降低，该螺旋包含与反核糖体结合位点（anti-RBS）配对的核糖体结合位点（RBS）。体外化学探测未观察到这种表达平台的调节，这表明拥挤的细胞环境并不妨碍检测紧密和松散的RNA调控构象。在此，我们描述了报告基因偶联（ReCo）icSHAPE的设计、方法、解释和注意事项。我们还描述了将差异ReCo-icSHAPE结果映射到Lrh核糖开关-前体Q共晶体结构上。该方法应易于应用于由效应物或环境变化触发的功能性RNA。
