Figure 4.

Queuosine is important for cytosolic codon-biased translation. (A) Schematic representation of dual luciferase construct in which Renilla fly luciferase (RLuc) is expressed in-frame with a codon-reengineered Firefly luciferase (FLuc), where the original sequence was left intact (WT), or all the Asn, Asp, Tyr and His codons were changed to NAC or NAU as indicated schematically in panel A. If Q has a role in translation of U-rich mRNAs, in absence of Q-tRNAs, NAU luciferase would be translated inefficiently, as depicted by the shortened grey arrow, leading to reduction in the levels of Firefly luciferase. Renilla luciferase levels, however, should remain comparable across different cell lines, as they are not subject to codon changes. (B) Number of cognate codons for Q-tRNAs in the three constructs. (C) Ratio of luminescence of FLuc to RLuc, further normalized to the WT construct. For this experiment, the cells were depleted for queuine for 48 h, and subsequently supplemented with 20 nM synthetic queuine (+ q) or vehicle control (− q) for 24 h. The graphs are representative of three independent experiments (* P <0.01, ns: not significant).