A robust method for measuring aminoacylation through tRNA-Seq

Abstract

Current methods to quantify the fraction of aminoacylated tRNAs, also known as the tRNA charge, are limited by issues with either low throughput, precision, and/or accuracy. Here, we present an optimized charge tRNA-Seq method that combines previous developments with newly described approaches to establish a protocol for precise and accurate tRNA charge measurements. We verify that this protocol provides robust quantification of tRNA aminoacylation and we provide an end-to-end method that scales to hundreds of samples including software for data processing. Additionally, we show that this method supports measurements of relative tRNA expression levels and can be used to infer tRNA modifications through reverse transcription misincorporations, thereby supporting multipurpose applications in tRNA biology.

A Translation-Independent Directed Evolution Strategy to Engineer Aminoacyl-tRNA Synthetases

“… 40 − 47 Recently, this strategy has been combined with modern enrichment and sequencing methods to perform such analyses in a more high-throughput manner. 42 , 43 , 45 − 47 After the selective periodate-mediated oxidation of the uncharged tRNAs and the deacylation of the charged tRNAs using mildly alkaline conditions, a unique oligonucleotide sequence can be introduced onto the intact 3′-terminus of the acylated population either through ligation 43 , 45 , 46 or by polymerase-mediated extension of the 3′-terminus using a DNA template that is hybridized onto the tRNA sequence. 44 The installed oligonucleotide sequence can be subsequently used to selectively reverse-transcribe and PCR amplify the charged tRNA sequences.…”

Eukaryotic AlaX provides multiple checkpoints for quality and quantity of aminoacyl-tRNAs in translation

“…In addition, with translation blocked (puromycin treatment), hydrolysis of Ser-tRNA Ser by hAlaX was more pronounced. Finally, two independent reports using the DM-tRNA-seq (demethylase-thermostable group II intron RT tRNA sequencing) method showed that the aminoacylation levels of tRNA Ser isoacceptors are the lowest among all human tRNAs ( 65 , 66 ). The rationale for the low levels of Ser-tRNA Ser compared with other tRNAs ( 65 , 66 ) is unclear.…”

“…Finally, two independent reports using the DM-tRNA-seq (demethylase-thermostable group II intron RT tRNA sequencing) method showed that the aminoacylation levels of tRNA Ser isoacceptors are the lowest among all human tRNAs ( 65 , 66 ). The rationale for the low levels of Ser-tRNA Ser compared with other tRNAs ( 65 , 66 ) is unclear. Whether hydrolysis of Ser-tRNA Ser by hAlaX is responsible for the low tRNA Ser charging levels in vivo requires further investigation.…”