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.”