Cytosolic Accumulation of Small Nucleolar RNAs (snoRNAs) is Dynamically Regulated by NADPH Oxidase

Small nucleolar RNAs (snoRNAs) guide nucleotide modifications of cellular RNAs in the nucleus. We have previously shown that box C/D snoRNAs from the Rpl13a locus are unexpected mediators of physiologic oxidative stress, independent of their predicted ribosomal RNA modifications. Here, we demonstrate that oxidative stress induced by doxorubicin causes rapid cytoplasmic accumulation of the Rpl13a snoRNAs through a mechanism that requires superoxide and a nuclear splice variant of NADPH oxidase. RNA-sequencing analysis reveals that box C/D snoRNAs as a class are present in the cytoplasm, where their levels are dynamically regulated by NADPH oxidase. These findings suggest that snoRNAs may orchestrate the response to environmental stress through molecular interactions outside of the nucleus. Cytosolic RNA was isolated from H9c2 cells by digitonin permeabilization of the plasma membrane and differential centrifugation. Cells were treated as follows: control, DPI (Nox inhibitor), doxorubicin (dox), or DPI+dox. N=3 independent experiments were analyzed for each treatment (12 samples total).

小核仁RNA（small nucleolar RNAs，snoRNAs）可在细胞核内介导细胞RNA的核苷酸修饰。本团队此前研究表明，源自Rpl13a基因座的C/D框小核仁RNA可作为生理性氧化应激的非预期介导因子，且不依赖于其预设的核糖体RNA修饰功能。本研究证实，阿霉素诱导的氧化应激可通过依赖超氧阴离子与烟酰胺腺嘌呤二核苷酸磷酸氧化酶（NADPH oxidase）核内剪接变体的机制，促使Rpl13a来源的snoRNAs快速在细胞质中积累。RNA测序（RNA-sequencing）分析显示，C/D框小核仁RNA作为一类整体存在于细胞质中，其表达水平由烟酰胺腺嘌呤二核苷酸磷酸氧化酶动态调控。上述研究结果表明，snoRNAs可通过细胞核外的分子相互作用，协调机体对环境应激的应答反应。本研究通过洋地黄皂苷对细胞膜进行透化处理结合差速离心法，从H9c2细胞中分离得到胞质RNA。细胞接受如下四种处理：空白对照组、DPI（Nox抑制剂）组、阿霉素（dox）组以及DPI+阿霉素联合处理组。每组处理均设置3次独立重复实验，共计12个样本用于数据分析。