Characterization of UVA-Induced Alterations to Transfer RNA Sequences

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.

RNA Cleavage Properties of Nucleobase-Specific RNase MC1 and Cusativin Are Determined by the Dinucleotide-Binding Interactions in the Enzyme-Active Site

“…A tertiary structure of RNase T1 (GenBank: RMZ48028.1) was generated by homology modeling and docked with a 3 -GMP ligand. This docked ligand exhibited identical overlay with the 3 -GMP bound T1 crystal structures-4GSP [27] or 1RGA [28] (Supplemental Figure S4) with high fitness score or solution validation parameters including PLP (Piecewise Linear Potential: 72), Gibbs free energy change (−20 kcal/mole) and high number (14) of hydrogen bonding interactions.…”

“…Simple atomic substitutions (C=O to C=S) or methylations make the modified uridine a non-substrate for MC1 [11]. The influence of chemical modifications on the cleavage property of these enzymes seems to depend on the altered chemical groups and interactions in the active site of protein tertiary structure [11][12][13][14][15][16]. While methylation of cytosine (m 5 C) does not affect its recognition as a substrate, RNA with tandem cytidines is not a substrate for cusativin [12], indicating differences in the binding site architecture (B1 and B2 sites) of these enzymes.…”

Oxidative Damage to RNA is Altered by the Presence of Interacting Proteins or Modified Nucleosides

“…It is interesting to note that although RlmKL installs methyl groups at two different positions (2069 and 2,445) of 23S rRNA compared to RlmB (at position 2,251) ( Popova and Williamson, 2014 ; Sergiev et al, 2018 ), the latter strain seems to be more susceptible to oxidative stress ( Figure 4 ). Further studies involving modification mapping experiments ( Sun et al, 2020 ; Thakur et al, 2020 ) could identify the exact locations in the sequence that might serve as hotspots for oxidative damage. Nevertheless, these studies indicate that although some post-transcriptional methylations are not essential for normal growth, their absence could aggravate the vulnerability of rRNA to oxidative damage.…”

“…The naturally occurring PTMs impart structural stability ( Decatur and Fournier, 2002 ; Väre et al, 2017 ), facilitate interactions between ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA) and translation factors ( Polikanov et al, 2015 ), and impart translation efficiency ( Agris et al, 2017 ). Our laboratory has previously documented oxidative changes to ribonucleosides and their PTMs in E. coli upon UVA-induced oxidative stress ( Sun et al, 2018 ) and these oxidative changes seem to localize at specific regions of tRNA ( Barciszewski et al, 1999 ; Sun et al, 2020 ). Here, we show that the oxidative changes in RNA are altered by the bound proteins and post-transcriptional nucleoside modifications such as methylations or the hypermodification, 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), found in ribosomal RNA and tRNA, respectively, of an E. coli model system.…”

Bacterial Response to Oxidative Stress and RNA Oxidation

“…An in vitro study where liquid chromatography tandem mass spectrometry was applied to E. coli tRNA showed that upon oxidation induced by UV radiation, several tRNA nucleosides were modified with the 8-oxo-G adduct ( Sun et al, 2018 ). Specifically, oxidative damage was mostly found to affect the anticodon and variable loop regions sequences of tRNA ( Sun et al, 2020 ), which correlates with translational errors in the decoding process and aminoacylation ( Yan and Zaher 2019 ). Zhong et al (2015) showed that under oxidative stress caused by H 2 O 2, the vast majority of tRNAs in E. coli were decreased in vivo .…”