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

RNA. 2020 Sep;26(9):1291-1298. doi: 10.1261/rna.075556.120. Epub 2020 May 21.

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: acid denaturing PAGE; galactosyl-queuosine; mannosyl-queuosine; queuosine.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Anticodon / chemistry
  • Anticodon / genetics
  • Cell Line, Tumor
  • Gels / chemistry*
  • Glycosylation
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • MCF-7 Cells
  • Nucleoside Q / chemistry*
  • Nucleoside Q / genetics
  • RNA, Transfer / chemistry*
  • RNA, Transfer / genetics*
  • Transfer RNA Aminoacylation / genetics

Substances

  • Anticodon
  • Gels
  • Nucleoside Q
  • RNA, Transfer