Processing of RNA Containing 8-Oxo-7,8-Dihydroguanosine (8-oxoG) by the Exoribonuclease Xrn-1.
Processing of RNA Containing 8-Oxo-7,8-Dihydroguanosine (8-oxoG) by the Exoribonuclease Xrn-1.
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DOI：
10.3389/fmolb.2021.780315
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发表时间：
2021
影响因子：
5
通讯作者：
Resendiz MJE
中科院分区：
文献类型：
--
作者：
Phillips CN;Schowe S;Langeberg CJ;Siddique N;Chapman EG;Resendiz MJE
由于氧化RNA与某些疾病的进展/发展以及衰老之间存在联系，因此了解细胞内氧化损伤RNA的处理方式具有重要意义。在负责修饰（化学或自然）RNA衰变的核糖核酸酶中，外切酶Xrn-1是一种催化5 ‘→3 ’方向磷酸化RNA水解的过程酶。我们开始探索这种外切酶对通过固相合成获得的含有8-氧-7,8-二氢鸟苷（8-oxoG）的RNA的寡核苷酸（20-nt至30-nt长）的反应性。结果表明，Xrn-1在含有8-oxoG的位点上停滞不前，通过电泳分析证明，与典型类似物相比，存在较慢的移动带。通过PAGE和MALDI-TOF对所观察到的片段进行了表征，证实该寡核苷酸片段含有一个5 ' -磷酸化的8-oxoG。此外，当8-oxoG位于不同位置和不同序列时，这种失速的产量在5% - 30%之间变化。为了更好地理解核酸酶效率降低的原因，我们探讨了：1)氢键和空间约束；2)反合成构象变化；3)二价阳离子浓度；4)二级结构。这是通过引入甲基化或溴化嘌呤（m1G, m6,6A或8-BrG），探测变化的[Mg2+]，并使用圆二色性（CD）来探索结构化RNA的形成来实现的。结果表明，糖苷键周围构象变化所施加的空间限制可能是造成失速的部分原因，然而，这些结果并不能完全解释某些观察到的较高失速收率。我们假设π-π堆积的改变以及8-oxoG与结合位点内残基之间诱导的h键相互作用也可能是Xrn-1效率降低的原因。总的来说，这些观察结果表明，其他尚未发现/确定的因素可能有助于氧化RNA的衰变。此外，Xrn-1降解含有m1G的RNA，并在遇到m6,6A或8-BrG的位点轻度停滞，这是特别有趣的，因为前两种是自然发生的修饰。
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.