Abstract
Ultraviolet radiation (UVR) adversely affects the integrity of DNA, RNA, and their nucleoside modifications. By employing liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based RNA modification mapping approaches, we identified the transfer RNA (tRNA) regions most vulnerable to photooxidation. Photooxidative damage to the anticodon and variable loop regions was consistently observed in both modified and unmodified sequences of tRNA upon UVA (λ 370 nm) exposure. The extent of oxidative damage measured in terms of oxidized guanosine, however, was higher in unmodified RNA compared to its modified version, suggesting an auxiliary role for nucleoside modifications. The type of oxidation product formed in the anticodon stem–loop region varied with the modification type, status, and whether the tRNA was inside or outside the cell during exposure. Oligonucleotide-based characterization of tRNA following UVA exposure also revealed the presence of novel photoproducts and stable intermediates not observed by nucleoside analysis alone. This approach provides sequence-specific information revealing potential hotspots for UVA-induced damage in tRNAs.
Highlights
Transfer RNAs deliver amino acids to the site of ribosome-mediated protein synthesis while decoding the messenger RNA
To identify the regions of transfer RNA (tRNA) that are vulnerable to photooxidation, both modified and unmodified tRNATyr were exposed to UVA
Since our main goal is to identify the effects of UVA on other nucleoside modifications, mapping of oxidation products was limited to the RNA that was exposed to UVA in the presence of riboflavin
Summary
Transfer RNAs (tRNAs) deliver amino acids to the site of ribosome-mediated protein synthesis while decoding the messenger RNA (mRNA) This critical functional role is facilitated by the highly conserved folding of tRNA, which is essential for recognition and charging by aminoacyl-tRNA synthetases, interactions with the translational apparatus, and codon–anticodon pairing [1,2,3,4,5]. Post-transcriptional nucleoside modifications (PTMs) are ubiquitous in tRNA ranging from single atom substitution to complex hypermodifications catalyzed by elaborate enzymatic pathways [6]. They present varied nucleoside chemistries ranging from hydrophobic (aliphatic chains or aromatic substituents) to hydrophilic (including charged) functional groups in the anticodon and its loop [7,8]. The chemical groups present elsewhere in the tRNA body (such as D-loop, TΨC-loop) provide functionalities for structural stabilization [9,10,11,12]
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