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Queuosine (Q) is a hypermodified base in the wobble anticodon position of tRNAs coding for the amino acids Tyr, His, Asn, and Asp. tRNA Q-modification is introduced by a queuine tRNA-ribosyltransferase (TGT) that replaces the guanine base at G34 at these tRNAs with the modified base. tRNA Q-modification is widely distributed among prokaryotic and eukaryotic organisms, but only bacteria synthesize Q-modified tRNA de novo. In mammals, tRNA Q-modifications strictly rely on the presence of gut microbiomes or diets to produce the queuine base. Despite decades of study, cellular roles of tRNA Q-modification are still not fully understood. Here we describe a method to quantify tRNA Q-modification levels in individual tRNAs from human cells based on the presence of a cis-diol in the Q modification. This cis-diol moiety slows modified tRNA migration through polyacrylamide gels supplemented with N-acryloyl-3-aminophenylboronic acid (APB) compared to the unmodified tRNA. This difference can be visualized by Northern blots using probes for specific tRNA.

Quantification of Queuosine Modification Levels in tRNA from Human Cells Using APB Gel and Northern Blot