Characterisation of EIF5A perturbations on ribosome dynamics with single-molecule imaging

The development of protein-tagging techniques such as SunTag has given unprecedented opportunities to measure translation dynamics in living cells. In this study we leveraged the SunTag system to investigate the regulatory role of translation elongation in protein synthesis. We focused on an important elongation factor, EIF5A, that relieves ribosome stalling in proline-rich sequences, such as collagen. We designed a SunTag reporter containing a subsequence of Collagen I alpha 1 (COL1A1), which is a putative target of EIF5A, together with other control reporters. We developed a Bayesian inference framework based on a low-density approximation of the Totally Asymmetric Exclusion Process (TASEP) to analyze both steady state and run-off assays and infer the kinetic parameters. Our results confirmed that translation occurs at low density in SunTag reporters exhibiting a wide range of initiation and elongation rates. This suggests a feedback mechanism where elongation influences initiation to prevent ribosome crowding on suboptimal sequences. By manipulating the levels of active EIF5A through GC7 treatment, we show that translation elongation was reduced on the SunTag-COL1A1 reporter, and on other reporters, with respect to normal conditions. The slow-down in elongation was accompanied by a similar decrease in initiation, leading to ribosome density conservation. Genetic knockout of EIF5A showed a stronger decrease in initiation than elongation, leading to a significant reduction in ribosome density that was not observed upon GC7 treatment, indicating distinct underlying mechanisms; steady-state trace analysis suggests that this effect may be due to altered bursting dynamics. The ubiquitous effects of EIF5A perturbation, not limited to the SunTag-COL1A1 reporter, implies a broader role of EIF5A in translation elongation, consistent with previous results in ribosome profiling data. Our findings extend the current knowledge on the interplay between elongation and initiation and propose new methodologies for analyzing SunTag traces, contributing to the broader understanding of translation regulation.