Characterisation of the cellular mechanisms involved in the transport and metabolism of the queuine micronutrient

Abstract

Queuine (q) is a modified derivative of the 7-deazaguanine nucleobase which is exclusively synthesized by bacteria and is salvaged by eukaryotic cells. Queuine is irreversibly exchanged with guanine 34 in the first position of the anticodon loop of tRNA that accept the amino acids for histidine, tyrosine, asparagine and aspartic acid. The queuine modification in tRNA, now a nucleoside – Queuosine (Q) is thought to modulate the rate and accuracy of mRNA translation. The insertion reaction into tRNA is performed by the queuine tRNA ribosyltransferase enzyme. The literature describes numerous other ill- defined enzymatic activities associated with queuine including one or more specific transporter(s), a salvage enzyme and one (or more) kinases. However, the identity of these proteins remains unknown. Post tRNA turnover, several possible queuosine precursors and phosphorylated derivatives – Q, 3'-QMP, 5'-QMP which could account for the salvage and recycling of the micronutrient back to q. Previously, the suspected salvage enzyme DUF2419 from S. pombe demonstrated weak in vitro Q to q conversion activity. Here we report biochemical evidence that HeLa cells harboring a knock-out of C9orf64 – the human ortholog of DUF2419 – fail to convert Q to q and instead accumulate a queuosine-5'- monophosphate (5'-QMP) intermediate. This shows that C9orf64 is a queuosine-nucleotide N-glycosylase (QNG1 in mammals). Moreover, we show that QNG1 is highly expressed in the liver and may be a focal point for Q salvage. The essential role this hydrolase may play in harvesting queuine in mammals may give further insight into the pathophysiological processes involved with queuine deficiency. Previously, literature has hinted at the existence of one or more transporter(s) involved in the uptake of q/Q in mammalian cells. Here we show in vitro evidence that SLC35F2 functions as the exclusive high affinity transporter for Q and the primary high affinity transporter for q. In the absence of SLC35F2, q import is substantially decreased (albeit not eliminated), hinting at the existence of a secondary low affinity transporter for q. By means of competitive uptake studies, SLC35F2 was found to displays high specificity for both q and Q as none of the canonical nucleobases nor nucleosides were capable of disrupting their import. The highly specific and essential role SLC35F2 plays in both q and Q transport, highlights the existence of yet another protein exclusive to the queuine pathway. The findings of this project clarify how eukaryotic organisms take up and salvage the queuine micronutrient. It is hoped the work will open new possibilities for understanding the role of queuine in the health and physiology of humans and other organisms.