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

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.