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

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...

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Published in:	Biochemistry (Easton) Vol. 56; no. 31; pp. 4029 - 4038
Main Authors:	Wulff, Thomas F, Argüello, Rafael J, Molina Jordàn, Marc, Roura Frigolé, Helena, Hauquier, Glenn, Filonava, Liudmila, Camacho, Noelia, Gatti, Evelina, Pierre, Philippe, Ribas de Pouplana, Lluís, Torres, Adrian G
Format:	Journal Article
Language:	English
Published:	United States American Chemical Society 08-08-2017
Subjects:	Adenosine - metabolism Adenosine Deaminase - chemistry Adenosine Deaminase - genetics Adenosine Deaminase - metabolism Amplified Fragment Length Polymorphism Analysis Base Pairing Computational Biology Deamination Expert Systems HeLa Cells Humans Hydrogen-Ion Concentration Inosine - metabolism Models, Biological Polymorphism, Restriction Fragment Length Reverse Transcription RNA Interference RNA Processing, Post-Transcriptional RNA, Small Interfering - metabolism RNA, Transfer, Ala - antagonists & inhibitors RNA, Transfer, Ala - metabolism RNA, Transfer, Thr - antagonists & inhibitors RNA, Transfer, Thr - metabolism RNA, Transfer, Val - antagonists & inhibitors RNA, Transfer, Val - metabolism Substrate Specificity
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Summary:	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 mRNAs. 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, amplified via polymerase chain reaction (PCR), and sequenced. ptRM-dependent base changes on reverse transcription 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 can reliably estimate the modification status of the sample, a feature that can be useful in the study of the regulatory role of tRNA modifications in gene expression.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0006-2960 1520-4995
DOI:	10.1021/acs.biochem.7b00324