A new method for identifying the amino acid attached to a particular RNA in the cell

Abstract

To investigate the function of tRNAs or any other aminoacylable RNAs in vivo, it is important to be able to estimate the amounts and species of aminoacylated RNAs in living cells. We have developed a method of analyzing amino acids attached to particular tRNAs obtained from cells. After the ester bond between the amino acid and the 3'-adenosine moiety of a specific aminoacyl-tRNA is stabilized by acetylation of the amino acid with [14C]acetic anhydride, the aminoacyl-tRNA can be fished out with a solid-phase-attached DNA probe. The t4C-labeled acetylamino acid is then released from the thus purified acetyl-aminoacyl-tRNAs by alkaline treatment and detected by TLC analysis.

Biogenesis of glutaminyl-mt tRNA Gln in human mitochondria

“…The isolation of individual species of aa-tRNA and analysis of the attached amino acids were conducted according to the method of Suzuki et al (23). Briefly, HeLa cells (7.5 ϫ 10 5 cells) were cultured and transfected with a mixture of three siRNAs targeting all subunits of hGatCAB (final concentration, 3.3 nM for each).…”

“…At 72 h post-transfection, total RNA was extracted from the cells, as described in SI Text. The amino groups of the amino acid moieties of the aa-tRNAs in the total RNA fraction were acetylated with acetic anhydride to stabilize the aminoacyl bond (23). The acetylaminoacyl-tRNAs were then isolated from the total RNA by a solid-phase DNA probe method using a 3Ј-biotinylated oligonucleotide with a sequence identical to that used for northern blotting, which was performed by immobilizing the oligonucleotide on streptavidin-agarose (GE Healthcare).…”

“…To obtain direct evidence that hGatCAB is a bona-fide Glu-AdT that can generate Gln-tRNA Gln in mitochondria, we devised a method to directly analyze amino acids attached to individual tRNAs isolated from cells (23). The method is outlined in Fig.…”

“…HeLa cells were harvested 72 h after transfection with three siRNAs targeting each of the hGatCAB subunits, and total RNA, including aa-tRNAs, was immediately extracted under acidic conditions at low temperature. To stabilize the aminoacyl-bond of each aa-tRNA, the ␣-amino group of the aminoacyl moiety was acetylated by acetic anhydride as described previously (23). The mt tRNA Gln was isolated using a solid-phase DNA probe method (15,23), and then completely digested by RNase I, yielding acetyl-aminoacyl-adenosines (Acaa-Ado) from the 3Ј-terminus of the tRNA.…”

“…To stabilize the aminoacyl-bond of each aa-tRNA, the ␣-amino group of the aminoacyl moiety was acetylated by acetic anhydride as described previously (23). The mt tRNA Gln was isolated using a solid-phase DNA probe method (15,23), and then completely digested by RNase I, yielding acetyl-aminoacyl-adenosines (Acaa-Ado) from the 3Ј-terminus of the tRNA. The Ac-aa-Ado species were determined by LC/MS analysis.…”