Detection and quantification of glycosylated queuosine modified tRNAs by acid denaturing and APB gels
- 1Department of Chemistry, University of Chicago, Chicago, Illinois 60637, USA
- 2Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310006, China
- 3Institute of Hematology, Zhejiang University, Zhejiang, 310006, China
- 4Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA
- 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.
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Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.075556.120.
- Received March 27, 2020.
- Accepted May 19, 2020.
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