No 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

Adenosine deaminase acting on transfer RNA (ADAT) is an essential eukaryotic enzyme that catalyzes the deamination of adenosine to inosine at the first position of tRNA anticodons. Mammalian ADATs modify eight different tRNAs, having increased their substrate range from a bacterial ancestor that likely deaminated exclusively tRNA Arg . Here we investigate the recognition mechanisms of tRNA Arg and tRNA Ala by human ADAT to shed light on the process of substrate expansion that took place during the evolution of the enzyme. We show that tRNA recognition by human ADAT does not depend on conserved identity elements, but on the overall structural features of tRNA. We find that ancestral-like interactions are conserved for tRNA Arg , while eukaryote-specific substrates use alternative mechanisms. These recognition studies show that human ADAT can be inhibited by tRNA fragments in vitro, including naturally occurring fragments involved in important regulatory pathways.