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 G₃₄U₃₅N₃₆ 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.

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

FIGURE 1.

Queuosine and glycosylated queuosine modification and acid denaturing gel/northern blot detection. (A) Structure of the protonated queuosine and glycosylated queuosine base. (B) Schematic of acid denaturing gel and northern blot detection of Q- and glycosylated Q-modified tRNAs. Northern blots can be performed using either ³²P-labeled or 5′ biotin-labeled tRNA probes. (C) Acid denaturing gel and northern blot showing separation of glycosylated Q-modified tRNA^Asp (mQ: mannosyl-Q) and tRNA^Tyr (gQ: galactosyl-Q) from unmodified tRNA (G) using ³²P labeled probes. Asterisk (*) indicates an unknown tRNA^Tyr species. 0Q: total RNA from cells without Q-modification; 100Q: total RNA from cells with full Q-modification. Total RNA was from HEK293T cells.

FIGURE 2.

Analysis of Q and glycosylated Q-modified tRNAs using biotinylated tRNA probes. (A) Acid denaturing gels and northern blot of individual, Q or glycosylated Q-modified tRNA. 0Q: total RNA from cells without Q-modification; 100Q: total RNA from cells with full Q-modification; 0Q:100Q: equal amount mixture of 0Q and 100Q total RNA. G: unmodified tRNA, Q: Q-modified tRNA, mQ: mannosyl-Q, gQ: galactosyl-Q. (B) Acid denaturing gels and northern blot of tRNA^Asp and tRNA^Tyr with (NaIO₄) and without (NaCl) periodate treatment. 0: total RNA from 0Q cells; 1, 24: total RNA from cells 1 and 24 h after the addition of queuine to 0Q cells. Asterisk (*) indicates an unknown tRNA^Tyr species. Total RNA was from MCF7 cells.

FIGURE 3.

Q and glycosylated Q-modification kinetics of HEK293T cells by APB and acid denaturing gels. (A) Time course of individual Q or glycosylated Q-modified tRNA. At t = 0, 1 µM queuine was added to the medium of 0Q cells, total RNA was extracted after designated time points in hours. tRNA^His and tRNA^Asn are from APB gels, tRNA^Tyr and tRNA^Asp are from acid denaturing gels. G: unmodified tRNA, Q: Q-modified tRNA, mQ: mannosyl-Q, gQ: galactosyl-Q. (B) Quantitative modification fraction over time for individual tRNAs, from 1 µM queuine data. For tRNA^His and tRNA^Asn, the modification fraction represents Q; for tRNA^Asp and tRNA^Tyr, the modification fraction represents the sum of the Q and glycosylated Q modifications. (C,D) Same as A,B except at t = 0, 10 nM queuine was added to the medium of 0Q cells.

FIGURE 4.

Q-modification kinetics of tRNA^Asp and tRNA^Tyr in HEK293T cells by APB gels. (A) At t = 0, 1 µM queuine was added to the medium of 0Q cells, total RNA was extracted after designated time points in hours. G: unmodified tRNA, Q: Q-modified tRNA, mQ: mannosyl-Q, gQ: galactosyl-Q. The reversal to fast migration in later time points corresponds to gQ or mQ products as indicated by the acid denaturing gel results from Figure 3. (B) Quantitative modification fraction over time for individual tRNAs, from 1 µM queuine data. For comparison the total modification fraction from acid denaturing gel results in Figure 3 are also shown. (C,D) Same as A,B except at t = 0, 10 nM queuine was added to the medium of 0Q cells.