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      Detection and quantification of glycosylated queuosine modified tRNAs by acid denaturing and APB gels

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          Abstract

          Queuosine (Q) is a conserved tRNA modification in bacteria and eukaryotes. Eukaryotic Q-tRNA modification occurs through replacing the guanine base with the scavenged metabolite queuine at the wobble position of tRNAs with G 34U 35N 36 anticodon (Tyr, His, Asn, Asp) by the QTRT1/QTRT2 heterodimeric enzyme encoded in the genome. In humans, Q-modification in tRNA Tyr and tRNA Asp are further glycosylated with galactose and mannose, respectively. Although galactosyl-Q (galQ) and mannosyl-Q (manQ) can be measured by LC/MS approaches, the difficulty of detecting and quantifying these modifications with low sample inputs has hindered their biological investigations. Here we describe a simple acid denaturing gel and nonradioactive northern blot method to detect and quantify the fraction of galQ/manQ-modified tRNA using just microgram amounts of total RNA. Our method relies on the secondary amine group of galQ/manQ becoming positively charged to slow their migration in acid denaturing gels commonly used for tRNA charging studies. We apply this method to determine the Q and galQ/manQ modification kinetics in three human cells lines. For Q-modification, tRNA Asp is modified the fastest, followed by tRNA His, tRNA Tyr, and tRNA Asn. Compared to Q-modification, glycosylation occurs at a much slower rate for tRNA Asp, but at a similar rate for tRNA Tyr. Our method enables easy access to study the function of these enigmatic tRNA modifications.

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          Most cited references22

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          Modifications and functional genomics of human transfer RNA

          Tao Pan (2018)
          Transfer RNA (tRNA) is present at tens of millions of transcripts in a human cell and is the most abundant RNA in moles among all cellular RNAs. tRNA is also the most extensively modified RNA with, on an average, 13 modifications per molecule. The primary function of tRNA as the adaptor of amino acids and the genetic code in protein synthesis is well known. tRNA modifications play multi-faceted roles in decoding and other cellular processes. The abundance, modification, and aminoacylation (charging) levels of tRNAs contribute to mRNA decoding in ways that reflect the cell type and its environment; however, how these factors work together to maximize translation efficiency remains to be understood. tRNAs also interact with many proteins not involved in translation and this may coordinate translation activity and other processes in the cell. This review focuses on the modifications and the functional genomics of human tRNA and discusses future perspectives on the explorations of human tRNA biology.
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            MODOMICS: a database of RNA modification pathways—2013 update

            MODOMICS is a database of RNA modifications that provides comprehensive information concerning the chemical structures of modified ribonucleosides, their biosynthetic pathways, RNA-modifying enzymes and location of modified residues in RNA sequences. In the current database version, accessible at http://modomics.genesilico.pl, we included new features: a census of human and yeast snoRNAs involved in RNA-guided RNA modification, a new section covering the 5′-end capping process, and a catalogue of ‘building blocks’ for chemical synthesis of a large variety of modified nucleosides. The MODOMICS collections of RNA modifications, RNA-modifying enzymes and modified RNAs have been also updated. A number of newly identified modified ribonucleosides and more than one hundred functionally and structurally characterized proteins from various organisms have been added. In the RNA sequences section, snRNAs and snoRNAs with experimentally mapped modified nucleosides have been added and the current collection of rRNA and tRNA sequences has been substantially enlarged. To facilitate literature searches, each record in MODOMICS has been cross-referenced to other databases and to selected key publications. New options for database searching and querying have been implemented, including a BLAST search of protein sequences and a PARALIGN search of the collected nucleic acid sequences.
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              The Queuine Micronutrient: Charting a Course from Microbe to Man

              Micronutrients from the diet and gut microbiota are essential to human health and wellbeing. Arguably, among the most intriguing and enigmatic of these micronutrients is queuine, an elaborate 7-deazaguanine derivative made exclusively by eubacteria and salvaged by animal, plant and fungal species. In eubacteria and eukaryotes, queuine is found as the sugar nucleotide queuosine within the anticodon loop of transfer RNA isoacceptors for the amino acids tyrosine, asparagine, aspartic acid and histidine. The physiological requirement for the ancient queuine molecule and queuosine modified transfer RNA has been the subject of varied scientific interrogations for over four decades, establishing relationships to development, proliferation, metabolism, cancer, and tyrosine biosynthesis in eukaryotes and to invasion and proliferation in pathogenic bacteria, in addition to ribosomal frameshifting in viruses. These varied effects may be rationalized by an important, if ill-defined, contribution to protein translation or may manifest from other presently unidentified mechanisms. This article will examine the current understanding of queuine uptake, tRNA incorporation and salvage by eukaryotic organisms and consider some of the physiological consequence arising from deficiency in this elusive and lesser-recognized micronutrient.
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                Author and article information

                Journal
                RNA
                RNA
                RNA
                RNA
                Cold Spring Harbor Laboratory Press
                1355-8382
                1469-9001
                September 2020
                : 26
                : 9
                : 1291-1298
                Affiliations
                [1 ]Department of Chemistry, University of Chicago, Chicago, Illinois 60637, USA
                [2 ]Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310006, China
                [3 ]Institute of Hematology, Zhejiang University, Zhejiang, 310006, China
                [4 ]Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA
                Author notes
                Corresponding author: taopan@ 123456uchicago.edu
                Author information
                http://orcid.org/0000-0001-8577-2613
                Article
                9509184 RA
                10.1261/rna.075556.120
                7430669
                32439717
                66cc03be-df6d-4a9a-ae5c-77b6b640ad9a
                © 2020 Zhang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

                This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

                History
                : 27 March 2020
                : 19 May 2020
                Page count
                Pages: 12
                Funding
                Funded by: Department of Defense , open-funder-registry 10.13039/100000005;
                Funded by: Congressionally Directed Medical Research Programs , open-funder-registry 10.13039/100000090;
                Funded by: DoD/CDMRP
                Award ID: BC160450
                Funded by: National Institute of General Medical Sciences (NIGMS) , open-funder-registry 10.13039/100000057;
                Award ID: R01GM113194
                Funded by: China Scholarship Council , open-funder-registry 10.13039/501100004543;
                Award ID: 201906320410
                Categories
                Method

                queuosine,mannosyl-queuosine,galactosyl-queuosine,acid denaturing page

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