(592) Ribosome profiling reveals distinct translation termination kinetics and efficiency between FRT and 16HBE cells

Background: It is generally assumed that translation machinery is highly conserved among a range of mammalian species and cell types, thereby allowing cross-utility in the screening of premature termination codon (PTC) readthrough modulators. However, we and others have observed a trend that efficacious hits from Fischer rat thyroid (FRT) cell models fail to translate to human bronchial epithelial-derived 16HBE cells. For example, the combination of SRI-37240 (eukaryotic release factor (eRF) 1 protein degrader) and G418 (aminoglycoside) is highly efficacious only in FRT [1]. Discrepancies in cell permeability, pharmacokinetics, and/or abundance of translation termination factors are thought to affect the efficacy. Moreover, in differential gene expression analysis on FRT and 16HBE cell baseline samples, we observed substantial decreases in gene expression of several ribosomal components in FRT, suggesting the ribosome heterogeneity may confer distinct translation termination efficiency and response to the PTC readthrough modulators.

Methods: We use ribosome profiling to examine differences in global translation events between FRT and 16HBE cells. Lysates were digested with RNase I, and ribosome-protected mRNA fragments (RPF) were isolated and sequenced. RPF sequencing reads were aligned to representative transcripts of each gene (Ensembl Genes 105; Human: Ensembl Canonical transcripts in GRCh38, Rat: APPRIS transcripts in mRatBN7). Global ribosome occupancy and eRF1 binding at stop codons, inferred by the number and size of RPFs, respectively, are measured only in transcripts with ribosome density in the coding sequence greater than 20 reads per kilobase of transcript per million reads (~6,000 transcripts).

Results: Results revealed noticeable differences in global ribosome occupancy between FRT and 16HBE are manifest in translation initiation and termination, but not elongation. Compared to 16HBE, FRT showed lower occupancy at the initiation stage, which is retained in the early stage of the elongation, whereas the occupancy was substantially higher at the termination stage. Given that ribosome occupancy inversely correlates with ribosomal translation kinetics, the results indicate the termination kinetics in FRT are relatively slower. Moreover, transcripts with RPFs in the 3’ untranslated regions (3’UTR) have significantly higher ribosome occupancy at the termination stage compared to transcripts without 3’UTR RPFs in both FRT and 16HBE, suggesting that slow termination kinetics are indicative of transcripts with a low termination efficiency. Lastly, we found no noticeable difference in 21-nt RPF distribution at the termination stage between the cell types. Considering that lowering of eRF1/3 binding to the ribosomal A-site at the termination stage (e.g. SRI-37240) induces higher distribution of 21-nt RPF (reflecting vacant ribosomal A-site), the result suggests post-eRF1/3 binding events (e.g. eRF1 accommodation) are largely responsible for the slow termination kinetics in FRT.

Conclusions: These results demonstrate FRT cells have markedly slower termination kinetics relative to that of 16HBE cells, which is likely reflected by the lower termination efficiency. Ribosome heterogeneity may be a critical factor influencing susceptibility to translation termination suppression.

Acknowledgements: Supported by CFF.

References: [1] Sharma J, Du M, Wong E, Mutyam V, Li Y, Chen J, et al. A small molecule that induces translational readthrough of CFTR nonsense mutations by eRF1 depletion. Nat Commun. 2021;12(1):4358.