Detection of a Subset of Posttranscriptional Transfer RNA Modifications in Vivo with a Restriction Fragment Length Polymorphism-Based Method

Abstract

Transfer RNAs (tRNAs) are among the most heavily modified RNA species. Posttranscriptional tRNA modifications (ptRMs) play fundamental roles in modulating tRNA structure and function, and are being increasingly linked to human physiology and disease. Detection of ptRMs is often challenging, expensive and laborious. Restriction Fragment Length Polymorphism (RFLP) analyses study the patterns of DNA cleavage after restriction enzyme treatment; and have been used for the qualitative detection of modified bases on messenger RNAs. It is known that some ptRMs induce specific and reproducible base 'mutations' when tRNAs are reverse transcribed. For example, inosine, which derives from the deamination of adenosine, is detected as a guanosine when an inosine-containing tRNA is reverse transcribed, PCR-amplified and sequenced. ptRMdependent base changes on RT-PCR amplicons generated as a consequence of the reverse transcription reaction might create or abolish endonuclease restriction sites. The suitability of RFLP for the detection and/or quantification of ptRMs has not been studied thus far. Here we show that different ptRMs can be detected at specific sites of different tRNA types by RFLP. For the examples studied we show that this approach is able to reliably estimate the modification status of the sample, a feature that can be useful in the study of the regulatory role of tRNA modifications on gene expression.3

Identification and rescue of a tRNA wobble inosine deficiency causing intellectual disability disorder

“…2B, Valine-AAC, children II:1 and II:2). The presence of the C peak is due to tRNA-Val-CAC, which is nearly identical to tRNA-Val-AAC and is recognized by the same oligos used for RT-PCR amplification (Wulff et al 2017). We also detected a similar decrease in inosine modification when examining the sequencing chromatograms of tRNA-Pro-AGG.…”

“…As additional verification for reduced wobble inosine modification, we monitored wobble inosine status in specific tRNAs using RT-PCR sequencing. Since inosine is a guanosine (G) analog, the presence of inosine can be detected by sequencing of cDNA products generated by RT-PCR of cellular tRNA (Motorin et al 2007;Kawahara 2012;Wulff et al 2017). Using this strategy, we measured the wobble inosine status of tRNA-Val-AAC and tRNA-Pro-AGG in the fibroblast cell lines.…”

Protein folding and tRNA biology

“…This procedure, by avoiding the retrotranscription of the anticodon, limits the identification of different isodecoders. Also, by RFLP (restriction fragment length polymorphism) studies, different post-transcriptional modifications at specific sites of different tRNA types have been detected, taking advantage of the fact that base changes on reverse transcription PCR amplicons generated as a consequence of posttranscriptional modifications might create or abolish endonuclease restriction sites (Wulff et al 2017).…”

tRNA deamination by ADAT requires substrate-specific recognition mechanisms and can be inhibited by tRFs

“…We then performed inhibition assays of ADAT-dependent deamination using tRNA fragments, to determine if the mode of tRNA recognition by ADAT is equivalent between human tRNA Arg ACG and tRNA Ala AGC . Using the RFLP method (Wulff et al 2017) we examined I34 formation in tRNA Arg ACG (3 µM) in the presence of 50 µM tRNA Ala AGC fragments, and vice versa (Fig. 4).…”