Background

Transfer RNAs (tRNA) contain nucleotide modifications that perform a wide range of roles in translation and in the generation of tRNA fragments that are functional small RNAs. Queuosine (Q, Figure 1) is a tRNA modification that occurs in the wobble anticodon position of tRNAs coding for amino acids Tyr, His, Asn, and Asp. All these tRNAs contain G³⁴U³⁵N³⁶ anticodon sequences. In bacteria, G34-to-Q34 modification can be synthesized de novo through a cascade of enzyme reactions (El Yacoubi et al., 2012). In mammals, the G34 base in these tRNAs can be replaced with queuine (Figure 1) derived from either gut microbiome or diet by a two-protein complex enzyme QTRT1 (catalytic subunit) and QTRT2 encoded in the host genome. Q is widely distributed among prokaryotic and eukaryotic organisms, but its cellular roles are still not fully understood (Rakovich et al., 2011; Fergus et al., 2015; Tuorto et al., 2018; Wang et al., 2018). For example, tRNA Q-modification levels have been correlated with cancer progression and cellular metabolism.

To understand the role of Q-tRNA modification in mammalian cells, we performed comparative studies of cells having various levels of Q-modification (Wang et al., 2018). We found that tRNAs in cultured cell lines are Q-modified at 5%-60% in standard culture medium. Using dialyzed serum medium which was deprived of small molecules like queuine and continued passage of cell divisions, tRNA Q-modification levels continuously decreased until they became undetectable (0Q cells). The addition of 0.1-1 µM queuine to the media restored Q-modification to 100% levels within 12-24 h (100Q cells). To measure the level of tRNA Q-modification in individual tRNA, we used a previously developed method on the basis of the structure of queuosine (Igloi and Kossel, 1985; Zaborske et al., 2014). Cis-diol moieties, such as the 3’ ribose of a tRNA, slow migration through polyacrylamide gels supplemented with N-acryloyl-3-aminophenylboronic acid (APB). Since queuosine has an additional ribose moiety, the migration of Q-modified tRNA in APB gel is slower compared to unmodified tRNA. This differential migration of Q-modified versus unmodified tRNA can be determined by Northern blot analysis using probes specific for each tRNA. The mammalian tRNA^Asp and tRNA^Tyr are further glycosylated at the cis-diol of the modified base with mannose (man-Q) or galactose (gal-Q) which prevents the reaction between the Q-base and APB; our approach therefore only works for tRNA^His and tRNA^Asn in mammalian cells. Using this method and other techniques, we showed that Q-modification protects cognate tRNAs against ribonuclease cleavage in vitro and in cells which alters the abundance of tRNA fragments (tRF) in human cells (Wang et al., 2018).

Previously, the Q-content in total tRNA in mammalian cells was evaluated by taking advantage of the irreversible nature of the N-glycosidic bond of the Q-base to the sugar-ribose backbone. Total tRNAs were isolated and the extent of Q-modified in total tRNA was measured by the exchange of ³H-guanine with the guanine base of unmodified tRNAs catalyzed by the E. coli TGT enzyme. This exchange reaction was blocked by the Q-modification of the same tRNAs. Total Q-content was estimated based on the fraction of tRNAs that did not show exchange. This approach was first used to determine the Q-modification content in mammalian tissues and bacterial cells during different stages of development and aging (Singhal et al., 1981). Although useful, this previous method could not measure Q-modification level of individual tRNA. In contrast, our APB-gel based method directly measures the Q-modification levels of individual tRNA species starting with as little as 1-2 µg of total RNA.


Figure 1. Queuosine (Q) tRNA modification in four tRNAs. Diagram of G34 to Q34 modifications in the wobble anticodon position of tRNA^{His/Asn/Asp/Tyr}. Queuine corresponds to the nucleobase of Q-nucleotide. QTRT1 and QTRT2 (formerly QTRTD1) proteins are encoded in the human genome. Black dots represent the tRNA body; the QTRT1/QTRT2 complex introduces Q modification in the place of G using queuine as substrate. Q34 in tRNA^Asp and tRNA^Tyr are further glycosylated at the cis-diol of the modified base with mannose (man-Q) or galactose (gal-Q) in mammals. Our method measures tRNA^His and tRNA^Asn, but not tRNA^Asp and tRNA^Tyr because of their glycosylation.