Feedback from the Nascent Chain Triggers Ribosomal Frameshifting and Transcript Decay
Description
Based off previous evidence of the nascent chain’s impact on viral recoding mechanisms during translation, we wanted to explore if this mechanism is more common than just the viral context. 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 transmembrane domain (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. The LepB constructs were then implemented into cellular frameshift reporters, and the upstream TMD distance relative to a slippery sequence was validated to impact -1 PRF efficiency. This data was collected via flow cytometry of cells transfected with the frameshift reporters. In these reporters, GFP will only be translated via a -1 frameshift event during translation of the protein of interest. mKate is translated off of an IRES as an expression control. A bioinformatic search was then carried out, searching the human transcriptome for motifs (termed TMD-slip motifs) that have a TMD 35-55 amino acids upstream of a predicted slippery sequence. This search identified thousands of transcripts that have TMD-slip motifs. A deep proteomic data set was then searched for predicted -1 PRF products based off the list of transcripts that have at least one TMD-slip motif, and the findings confirmed evidence of -1 PRF arising from translation of TMD-slip motifs. We then used a recently published NMD substrate list to assess if our list of transcripts bearing TMD-slip motifs are enriched within it. We found that the TMD-slip containing transcripts are indeed enriched in NMD substrates upregulated upon UPF1 (NMD regulator) depletion. A splice variant of a voltage-gated ion channel (KCNQ1) was identified to have a TMD-slip motif that is absent in the canonical transcript. Using cellular reporters, the -1 PRF efficiencies were measured for both KCNQ1 transcripts. It was found that the -1 PRF efficiency for the splice variant was 8-fold larger than that of the canonical transcript. This data was obtained as described above using flow cytometry. This data set comprises of Figures 1, 2, 3, 6, 7, S2, S3 and Tables S1 and S2 of the paper titled “Feedback from the Nascent Chain Triggers Ribosomal Frameshifting and Transcript Decay.”
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Institutions
- Indiana University Bloomington
- Purdue University