Abstract
Understanding how oxidatively damaged RNA is handled intracellularly is of relevance due to the link between oxidized RNA and the progression/development of some diseases as well as aging. Among the ribonucleases responsible for the decay of modified (chemically or naturally) RNA is the exonuclease Xrn-1, a processive enzyme that catalyzes the hydrolysis of 5′-phosphorylated RNA in a 5′→3′ direction. We set out to explore the reactivity of this exonuclease towards oligonucleotides (ONs, 20-nt to 30-nt long) of RNA containing 8-oxo-7,8-dihydroguanosine (8-oxoG), obtained via solid-phase synthesis. The results show that Xrn-1 stalled at sites containing 8-oxoG, evidenced by the presence of a slower moving band (via electrophoretic analyses) than that observed for the canonical analogue. The observed fragment(s) were characterized via PAGE and MALDI-TOF to confirm that the oligonucleotide fragment(s) contained a 5′-phosphorylated 8-oxoG. Furthermore, the yields for this stalling varied from app. 5–30% with 8-oxoG located at different positions and in different sequences. To gain a better understanding of the decreased nuclease efficiency, we probed: 1) H-bonding and spatial constraints; 2) anti-syn conformational changes; 3) concentration of divalent cation; and 4) secondary structure. This was carried out by introducing methylated or brominated purines (m1G, m6,6A, or 8-BrG), probing varying [Mg2+], and using circular dichroism (CD) to explore the formation of structured RNA. It was determined that spatial constraints imposed by conformational changes around the glycosidic bond may be partially responsible for stalling, however, the results do not fully explain some of the observed higher stalling yields. We hypothesize that altered π-π stacking along with induced H-bonding interactions between 8-oxoG and residues within the binding site may also play a role in the decreased Xrn-1 efficiency. Overall, these observations suggest that other factors, yet to be discovered/established, are likely to contribute to the decay of oxidized RNA. In addition, Xrn-1 degraded RNA containing m1G, and stalled mildly at sites where it encountered m6,6A, or 8-BrG, which is of particular interest given that the former two are naturally occurring modifications.
Highlights
Oxidative stress occurs from an imbalance between the generation of reactive oxygen species (ROS) and the biological mechanisms naturally designed to cope with the same
In this study we found that the presence of 8-oxoG within RNA presents a challenge for Xrn-1 and stalls its processive degradation in ratios that varied on the position of the oxidative lesion within the oligonucleotide, as well as its sequence
The canonical strand 1′′ resulted in a suitable Xrn-1 substrate, with efficient degradation, we considered two aspects that led us to probe for RNA structure; 1) that Xrn-1 induced degradation of RNA is halted upon encountering highly structured RNA; (Wastika et al, 2020) and 2) that the presence of one 8-oxoG lesion can result in structural changes even when using short oligonucleotides of RNA (Choi et al, 2017)
Summary
Oxidative stress occurs from an imbalance between the generation of reactive oxygen species (ROS) and the biological mechanisms naturally designed to cope with the same. One of the main outcomes from such reactions leads to the generation of 8oxo-7,8-dihydroguanosine (8-oxoG), a common biomarker for oxidative stress (Christensen et al, 2018; Rodríguez-Callejas et al, 2019) These aspects bring into question the factors, mechanisms, and biological pathways that have evolved to handle oxidative damage of RNA. Another study showed that Xrn-1 (along with Rat1) play an important role in the degradation of mature, hypomodified, tRNA, (Chernyakov et al, 2008) highlighting its ability to process and degrade RNA containing various modifications. We considered reports from our group that describe differences in the way oxidatively damaged RNA reacts with RNases, as well as structural differences that arise from the presence of these type of lesions (Herbert et al, 2018) This prompted us to explore the degradation of oxidatively damaged RNA by Xrn-1, using oligonucleotides of RNA containing 8-oxoG as models
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