Quantification of Queuosine Modification Levels in tRNA from Human Cells Using APB Gel and Northern Blot
- 1 January 2019
- journal article
- research article
- Published by Bio-Protocol, LLC in Bio-Protocol
- Vol. 9 (6)
- https://doi.org/10.21769/bioprotoc.3191
Abstract
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.Keywords
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