tRNA modifications during translation : the roles of dihydrouridine and queuosine

Abstract

The high and still increasing variety of tRNA modifications is crucial for all roles tRNA is playing in biological systems. tRNA modifications have been shown to modulate translational processes, thereby regulating gene expression, especially as reaction to stress stimuli. The dihydrouridine (D) modification affects tRNA flexibility and is thus thought to play an important role in the overall tRNA structure and function. However, it is still largely unknown whether D is involved in translation and whether the NADPH-dependent synthesis of D is affected by stress stimuli. Here, the intracellular redox state in Escherichia coli (E. coli) was altered using the bipyridine salt paraquat which resulted in different sensitivities of the three tRNA-dihydrouridine synthases (Dus) towards oxidative stress. The activities of DusB and DusC were severely affected by paraquat, whereas DusA was highly active even under paraquat treatment. The analysis of E. coli polysome preparations did not reveal an indication that D plays a specific role in translation, although D-modified tRNAs were found on actively translating polysomes. Whereas D is a rather simple structured modification of the nucleobase, another project focused on the hypermodification queuosine (Q), which is located at the wobble position in the tRNA anticodon loop and was reported to modulate mRNA translation. In this work, an increase of Q modifications on tRNAs was detected in E. coli polysome preparations in comparison to the cytosolic tRNA pool, confirming that Q is playing a role in the translational process. Further, a non-natural azide-containing analogue of the precursor preQ1 was efficiently incorporated into tRNA via transglycosylation in vitro and in vivo in E. coli, Schizosaccharomyces pombe (S. pombe) and human cells. The resulting semi-synthetic modification, termed Q L1, was also present in tRNAs on actively translating ribosomes, indicating the functional integration into translational processes. Q-L1 was also functionally integrated into an RNA modification circuit in S. pombe, replacing the natural Q in its stimulation of further methylation of cytosine 38 in tRNAAsp. This functional and minimally invasive replacement of Q with non-natural derivatives enables the incorporation of clickable conjugates in RNA in vivo and opens up for new opportunities investigating the biological role of Q.