Plant, Animal, and Fungal Micronutrient Queuosine Is Salvaged by Members of the DUF2419 Protein Family

Abstract

Queuosine (Q) is a modification found at the wobble position of tRNAs with GUN anticodons. Although Q is present in most eukaryotes and bacteria, only bacteria can synthesize Q de novo. Eukaryotes acquire queuine (q), the free base of Q, from diet and/or microflora, making q an important but under-recognized micronutrient for plants, animals, and fungi. Eukaryotic type tRNA-guanine transglycosylases (eTGTs) are composed of a catalytic subunit (QTRT1) and a homologous accessory subunit (QTRTD1) forming a complex that catalyzes q insertion into target tRNAs. Phylogenetic analysis of eTGT subunits revealed a patchy distribution pattern in which gene losses occurred independently in different clades. Searches for genes co-distributing with eTGT family members identified DUF2419 as a potential Q salvage protein family. This prediction was experimentally validated in Schizosaccharomyces pombe by confirming that Q was present by analyzing tRNAAsp with anticodon GUC purified from wild-type cells and by showing that Q was absent from strains carrying deletions in the QTRT1 or DUF2419 encoding genes. DUF2419 proteins occur in most Eukarya with a few possible cases of horizontal gene transfer to bacteria. The universality of the DUF2419 function was confirmed by complementing the S. pombe mutant with the Zea mays (maize), human, and Sphaerobacter thermophilus homologues. The enzymatic function of this family is yet to be determined, but structural similarity with DNA glycosidases suggests a ribonucleoside hydrolase activity.

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

“…The digestion product of most interest for this work was that of the anticodon of the tRNA with the sequence CCU[Q]UCA[m 5 C]Gp. 46 This oligonucleotide was found to elute at 39.0 min. with other nearly co-eluting oligonucleotides.…”

“…For instance, RNase T1 digests on the 3' end of unmodified guanosine, 45 and has been found to be inhibited by N 2 -methylguanosine (m 2 G) in certain instances. 46 By limiting the number of guanosines to one in a composition, the number of compositions to be evaluated against the experimental data is reduced. With this limited number of potential compositions, the resulting MS/MS data can be analyzed to determine the composition that is correct and the order of the nucleotides in the sequence.…”

“…The procedure is also repeated with other RNase digestions, if necessary, to ensure appropriate sequence coverage of the RNA of interest. 46 Most commonly, the RNA modification mapping approach has been used for the placement of modified nucleosides onto a single RNA sequence, although large numbers of smaller RNA sequences or very large (e.g., rRNA) sequences can be analyzed in a serial fashion. For example, the Suzuki group used their Chaplet chromatography system 49 to isolate each bovine mitochondrial tRNA for LC-MS/MS analysis of both the nucleosides and oligonucleotide digests.…”

Genome recoding by tRNA modifications

“…This complex consists of the catalytic subunit, Q-tRNA-ribosyltransferase 1 (Qtrt1), and a homologous accessory subunit, Q-tRNA-ribosyltransferase domain containing 1 (Qtrtd1). The complex incorporates queuine into cytosolic tRNA-Tyr, -Asn, -Asp and -His, and into mitochondrial tRNA-Asp [34]. tRNA-Asp and tRNA-Tyr are further modified to mannosyl Q-tRNA (manQ34) and galactosyl Q-tRNA (galQ34), respectively [35].…”

Cofactor binding determinants in the NADPH-dependent nitrile-oxidoreductase QueF

“…tRNA effectively translates genetic information to peptide by matching the correct amino acid to the genetic information carried by the mRNA in the ribosome 1 found in tRNAs with the GUN anticodon consensus sequence (tRNA asp , tRNA asn , tRNA his , tRNA tyr ) and serves as a structurally constraining base for tRNA codon loop flexibility. Queuosine is an essential micronutrient obtained by eukaryotes from diet or gut bacteria 5,6 .…”