Processing of RNA Containing 8-Oxo-7,8-Dihydroguanosine (8-oxoG) by the Exoribonuclease Xrn-1

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.

Dynamic proximity interaction profiling suggests that YPEL2 is involved in cellular stress surveillance

“…For instance, XRN1, one of the YPEL2 proximity partners, is a member of the 5′→3′‐exoribonucleases family that plays critical roles in mRNA processing and turnover, no‐go and nonsense‐mediated decay, as well as the RNA interference pathways. It is shown that o 8 G modifications induce translation stalling (Simms et al, 2014 ) and decrease the processing efficiency of XRN1 (Phillips et al, 2021 ). In addition, ribosome rescue upon translation stalling is carried out by a set of proteins, including ABCE1, a proximity interacting partner of YPEL2, which participates in the alleviation of stalling‐induced translational stresses (Pisareva et al, 2011 ).…”

8-Oxoguanine: from oxidative damage to epigenetic and epitranscriptional modification

“…o 8 G-mRNA accumulates in the absence of NGD factors in yeasts (Dom34p and Xrn1p) 142 . Notably, recent in vitro assays showed that Xrn1 stalls at the o 8 G sites, suggesting the presence of other factors that contribute to the decay of oxidized RNA 145 . Concomitantly, the associated ribosome quality control is activated, depending on LTN1 and Hel2 expression with oxidation and alkylation damage agents in yeasts 146 .…”

Dynamic proximity interaction profiling suggests that YPEL2 is involved in cellular stress surveillance

“…For instance, XRN1, one of the YPEL2 proximity partners, is a member of the 5′→3′-exoribonucleases family that plays key roles in mRNA processing and turnover, no-go and nonsense-mediated decay, as well as the RNA interference pathways 144 . It is shown that o 8 G modifications induce translation stalling 145 and decrease the processing efficiency of XRN1 146 . In addition, ribosome rescue upon translation stalling is carried out by a set of proteins including ABCE1, a proximity interacting partner of YPEL2, which participates in the alleviation of stalling-induced translational stresses 147 .…”