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

  1. Tao Pan4
  1. 1Department of Chemistry, University of Chicago, Chicago, Illinois 60637, USA
  2. 2Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310006, China
  3. 3Institute of Hematology, Zhejiang University, Zhejiang, 310006, China
  4. 4Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA
  1. Corresponding author: taopan{at}uchicago.edu

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 G34U35N36 anticodon (Tyr, His, Asn, Asp) by the QTRT1/QTRT2 heterodimeric enzyme encoded in the genome. In humans, Q-modification in tRNATyr and tRNAAsp 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, tRNAAsp is modified the fastest, followed by tRNAHis, tRNATyr, and tRNAAsn. Compared to Q-modification, glycosylation occurs at a much slower rate for tRNAAsp, but at a similar rate for tRNATyr. Our method enables easy access to study the function of these enigmatic tRNA modifications.

Keywords

  • Received March 27, 2020.
  • Accepted May 19, 2020.

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/.

| Table of Contents