Key result
EMCV IRES-mediated translation sites recruit two to three times fewer ribosomes than Cap-dependent translation sites under normal conditions, though the balance shifts in favor of IRES during cellular stress.
Why the study?
While viral hijacking of host translation machinery via IRES sequences has been studied in bulk assays, the dynamics at the single-molecule level remained unexplored in living cells.
Bicistronic biosensors combined with single-molecule tracking reveal that IRES-mediated translation recruits fewer ribosomes than Cap-dependent translation under normal conditions, but is more robust during cellular stress.
Caution against clinical extrapolation from cell models; leaves open IRES relevance in stressed cardiomyocytes.
Viruses use IRES sequences within their RNA to hijack translation machinery and thereby rapidly replicate in host cells. While this process has been extensively studied in bulk assays, the dynamics of hijacking at the single-molecule level remain unexplored in living cells. To achieve this, we developed a bicistronic biosensor encoding complementary repeat epitopes in two ORFs, one translated in a Cap-dependent manner and the other translated in an IRES-mediated manner. Using a pair of complementary probes that bind the epitopes co-translationally, our biosensor lights up in different colors depending on which ORF is being translated. In combination with single-molecule tracking and computational modeling, we measured the relative kinetics of Cap versus IRES translation and show: (1) Two non-overlapping ORFs can be simultaneously translated within a single mRNA; (2) EMCV IRES-mediated translation sites recruit ribosomes less efficiently than Cap-dependent translation sites but are otherwise nearly indistinguishable, having similar mobilities, sizes, spatial distributions, and ribosomal initiation and elongation rates; (3) Both Cap-dependent and IRES-mediated ribosomes tend to stretch out translation sites; (4) Although the IRES recruits two to three times fewer ribosomes than the Cap in normal conditions, the balance shifts dramatically in favor of the IRES during oxidative and ER stresses that mimic viral infection; and (5) Translation of the IRES is enhanced by translation of the Cap, demonstrating upstream translation can positively impact the downstream translation of a non-overlapping ORF. With the ability to simultaneously quantify two distinct translation mechanisms in physiologically relevant live-cell environments, we anticipate bicistronic biosensors like the one we developed here will become powerful new tools to dissect both canonical and non-canonical translation dynamics with single-molecule precision.
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Koch et al. (2020) studied this question. Bicistronic biosensor vs. Cap-dependent translation vs IRES-mediated translation was evaluated on Relative kinetics and ribosome recruitment of Cap versus IRES translation. EMCV IRES-mediated translation sites recruit two to three times fewer ribosomes than Cap-dependent translation sites under normal conditions, though the balance shifts in favor of IRES during cellular stress.
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