Isoacceptor specific characterization of tRNA aminoacylation and misacylation in vivo

Abstract

Amino acid misincorporation during protein synthesis occurs due to misacylation of tRNAs or defects in decoding at the ribosome. While misincorporation of amino acids has been observed in a variety of contexts, less work has been done to directly assess the extent to which specific tRNAs are misacylated in vivo, and the identity of the misacylated amino acid moiety. Here we describe tRNA isoacceptor specific aminoacylation profiling (ISAP), a method to identify and quantify the amino acids attached to a tRNA species in vivo. ISAP allows compilation of aminoacylation profiles for specific isoacceptors tRNAs. To demonstrate the efficacy and broad applicability of ISAP, tRNAPhe and tRNATyr species were isolated from total aminoacyl-tRNA extracted from both yeast and Escherichia coli. Isolated aminoacyl-tRNAs were washed until free of detectable unbound amino acid and subsequently deacylated. Free amino acids from the deacylated fraction were then identified and quantified by mass spectrometry. Using ISAP allowed quantification of the effects of quality control on the accumulation of misacylated tRNA species under different growth conditions.

Translational fidelity and mistranslation in the cellular response to stress

“…Recent work has started to provide data to support mechanistic models for the potential benefits of adaptive mistranslation, but to date these studies have been confined to individual mistranslation events. Recent technical advances now provide the opportunity to substantially broaden our understanding of the role of mistranslation by allowing measurement of the rates of both misaminoacylation and mistranslation in vivo 117,118 . Such accurate error rate measurements will provide a means to properly establish the relevance of mistranslation in physiological contexts by delineating how the level of mistranslation correlates with a cell’s ability to adapt, survive and thrive under different conditions.…”

mRNA Translation Gone Awry: Translation Fidelity and Neurological Disease

“…These assays only monitor a fraction of the possible translational errors, and the development of unbiased approaches such as ribosome profiling has greatly enhanced our knowledge of the fundamental process of ribosome decoding. More recently, groups have developed techniques to examine global tRNA aminoacylation and misaminoacylation in vivo , approaches that will be beneficial for study of diseases caused by mutations in the tRNA synthetases [77,78]. Furthermore, changes in the proteome resulting from mistranslation have begun to be assessed using sophisticated mass spectrometry techniques [79,80].…”

Editing of misaminoacylated tRNA controls the sensitivity of amino acid stress responses in Saccharomyces cerevisiae

“…Aa-tRNA Phe was deacylated by incubating the SA-PMPs in 20 mM ammonium formate, pH, 10 at 37°C for 1 h. Following deacylation, SA-PMPs were removed using a magnetic pipet. The supernatant was dried down and amino acids were quantified via mass spectrometry, as described previously ( 26 ).…”