In this study, we demonstrate that 5S ribosomal RNA (rRNA), a highly structured and protein-bound RNA, is quite difficult to reduce with antisense oligonucleotides (ASOs). However, we found a single accessible site that was targetable with a high-affinity complementary ASO. The ASO appeared to bind to the site, recruit RNaseH1, and cause degradation of the 5S RNA. Intriguingly, we also observed that the same ASO induced an accumulation of pre-5S RNA, which may contribute to reduced levels of mature 5S rRNA. As expected, ASO mediated reduction of 5S RNA, and modest inhibition of processing of pre-5S RNA resulted in nucleolar toxicity. However, the toxicity induced was minimal compared with actinomycin D, consistent with its modest effects on pre-5S rRNA. Mechanistically, we show that the accumulation of pre-5S rRNA required ASO hybridization to the cognate rRNA sequence but was independent of RNaseH1 activity. We found that Ro60 and La, proteins known to bind misprocessed RNAs, likely sequester the ASO-pre-5S rRNA species and block RNaseH1 activity, thus identifying another example of competitive mechanisms mediated by proteins that compete with RNaseH1 for binding to ASO-RNA heteroduplexes.

中文翻译：

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靶向 5S 核糖体 RNA 的 Gapmer 反义寡核苷酸可以通过两种机制减少成熟的 5S 核糖体 RNA。

在这项研究中，我们证明了 5S 核糖体 RNA (rRNA)，一种高度结构化和蛋白质结合的 RNA，很难用反义寡核苷酸 (ASO) 减少。然而，我们发现了一个具有高亲和力互补 ASO 可定位的可访问站点。ASO 似乎与该位点结合，募集 RNaseH1，并导致 5S RNA 降解。有趣的是，我们还观察到相同的 ASO 诱导了 pre-5S RNA 的积累，这可能有助于降低成熟 5S rRNA 的水平。正如预期的那样，ASO 介导的 5S RNA 减少和对 pre-5S RNA 加工的适度抑制导致核仁毒性。然而，与放线菌素 D 相比，诱导的毒性很小，与其对 pre-5S rRNA 的适度影响一致。从机制上讲，我们表明，pre-5S rRNA 的积累需要与同源 rRNA 序列进行 ASO 杂交，但与 RNaseH1 活性无关。我们发现 Ro60 和 La，已知结合错误加工的 RNA 的蛋白质，可能隔离 ASO-pre-5S rRNA 种类并阻断 RNaseH1 活性，从而确定了由与 RNaseH1 竞争结合 ASO-RNA 异源双链体的蛋白质介导的竞争机制的另一个例子.