nucleoside-q and metaperiodate

Queuosine (Q) is a conserved tRNA modification at the wobble anticodon position of tRNAs that read the codons of amino acids Tyr, His, Asn, and Asp. Q-modification in tRNA plays important roles in the regulation of translation efficiency and fidelity. Queuosine tRNA modification is synthesized de novo in bacteria, whereas in mammals the substrate for Q-modification in tRNA is queuine, the catabolic product of the Q-base of gut bacteria. This gut microbiome dependent tRNA modification may play pivotal roles in translational regulation in different cellular contexts, but extensive studies of Q-modification biology are hindered by the lack of high throughput sequencing methods for its detection and quantitation. Here, we describe a periodate-treatment method that enables single base resolution profiling of Q-modification in tRNAs by Nextgen sequencing from biological RNA samples. Periodate oxidizes the Q-base, which results in specific deletion signatures in the RNA-seq data. Unexpectedly, we found that periodate-treatment also enables the detection of several 2-thio-modifications including τm5s2U, mcm5s2U, cmnm5s2U, and s2C by sequencing in human and E. coli tRNA. We term this method periodate-dependent analysis of queuosine and sulfur modification sequencing (PAQS-seq). We assess Q- and 2-thio-modifications at the tRNA isodecoder level, and 2-thio modification changes in stress response. PAQS-seq should be widely applicable in the biological studies of Q- and 2-thio-modifications in mammalian and microbial tRNAs.

Topics: Amino Acids; Animals; Anticodon; Escherichia coli; High-Throughput Nucleotide Sequencing; Humans; Mammals; Nucleoside Q; Periodic Acid; RNA, Transfer; Sulfur

Escherichia coli encodes YadB, a protein displaying 34% identity with the catalytic core of glutamyl-tRNA synthetase but lacking the anticodon-binding domain. We show that YadB is a tRNA modifying enzyme that evidently glutamylates the queuosine residue, a modified nucleoside at the wobble position of the tRNA(Asp) QUC anticodon. This conclusion is supported by a variety of biochemical data and by the inability of the enzyme to glutamylate tRNA(Asp) isolated from an E.coli tRNA-guanosine transglycosylase minus strain deprived of the capacity to exchange guanosine 34 with queuosine. Structural mimicry between the tRNA(Asp) anticodon stem and the tRNA(Glu) amino acid acceptor stem in prokaryotes encoding YadB proteins indicates that the function of these tRNA modifying enzymes, which we rename glutamyl-Q tRNA(Asp) synthetases, is conserved among prokaryotes.

Topics: Acylation; Anticodon; Base Sequence; Biological Evolution; Conserved Sequence; Escherichia coli; Glutamate-tRNA Ligase; Molecular Mimicry; Nucleoside Q; Periodic Acid; RNA, Bacterial; RNA, Transfer, Asp; RNA, Transfer, Glu