Nascent Chain-Mediated Ribosomal Frameshifting During Membrane Protein Translation

Based off previous evidence of the nascent chain’s impact on viral recoding mechanisms during translation, we wanted to explore if this kind of mechanism could be more generalized. In vitro translation of reporters containing slippery sequences at different positions within a model membrane protein (LepB) was supplemented with EasyTag L-35S labeled Methionine. Translation products were separated via SDS PAGE and imaged using phosphorimaging. The imaged gels were integrated to quantify the relative abundances of full length and truncated (frameshifted) products. It was observed that translocation of a TMD sequence located 45 amino acids upstream of the slippery sequence induced increased -1 PRF efficiencies relative to insertion of TMD sequences at 35 and 55 amino acids upstream. Using cellular bicistronic -1PRF reporters, we wanted to see if this kind of motif is found in human membrane proteins. In these reporters, GFP will only be translated via a -1 frameshift event during translation of the protein of interest. An mKate is translated off an IRES as an expression control. The cellular reporters were transfected into HEK293T cells and standard mammalian cell culture protocols were utilized. Data obtained from in vivo experiments was collected via flow cytometry, and controls for the -1 PRF reporters were utilized to validate the frameshift signal. To ensure that the changes in GFP were being observed in cells expressing the reporters at similar expression levels, analysis of the flow cytometry data was restricted to cells that have mKate intensities that fall within two standard deviations of the overall average of the median mKate fluorescent intensities. -1 PRF efficiencies were calculated dividing the GFP/mKate ratio of a reporter by the GFP/mKate ratio of a 0-frame control, where the GFP is translated in the same reading frame as the protein of interest, then multiplying that by 100%. This data set comprises of Figures 1, 2, 3, 5, 6, S2, S3, S4, and Tables 1 and S1 of the paper titled “Nascent Chain-Mediated Ribosomal Frameshifting During Membrane Protein Translation.”

基于此前关于新生肽链（nascent chain）对翻译过程中病毒重编码机制影响的研究证据，我们旨在探究此类机制是否具备更广泛的普适性。我们对包含滑动序列的报告基因（reporter）进行体外翻译，该滑动序列位于模型膜蛋白（LepB）的不同位置，反应体系中添加了EasyTag L-35S标记的甲硫氨酸。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离翻译产物，并通过磷屏成像进行检测。对成像后的凝胶进行灰度积分，以量化全长产物与截短（移码）产物的相对丰度。研究发现，相较于在滑动序列上游35或55个氨基酸处插入跨膜结构域（transmembrane domain, TMD）序列，在滑动序列上游45个氨基酸处的跨膜结构域序列转位可提升-1程序性核糖体移码（-1 programmed ribosomal frameshift, -1 PRF）效率。为探究此类移码基序是否存在于人类膜蛋白中，我们使用了细胞双顺反子-1PRF报告基因。在该类报告基因中，绿色荧光蛋白（green fluorescent protein, GFP）仅能在目的蛋白的翻译过程中通过-1移码事件完成翻译；而mKate则通过内部核糖体进入位点（internal ribosome entry site, IRES）进行翻译，作为表达水平对照。将该细胞报告基因转染至HEK293T细胞，并采用标准哺乳动物细胞培养方案进行培养。通过流式细胞术收集体内实验获得的相关数据，并以-1 PRF报告基因的对照样本验证移码信号。为确保仅分析表达水平相近的转染细胞，我们将流式细胞术数据的分析范围限定为：mKate荧光强度处于整体中位mKate荧光强度平均值±2个标准差区间内的细胞。-1 PRF效率的计算方式为：将报告基因的GFP/mKate比值除以零读框对照的GFP/mKate比值（其中零读框对照的GFP与目的蛋白处于同一读框），再乘以100%。本数据集包含题为《膜蛋白翻译过程中新生肽链介导的核糖体移码》的论文中的图1、图2、图3、图5、图6、补充图S2、补充图S3、补充图S4以及表1和补充表S1。