Department of Core Antisense Research, Ionis Pharmaceutics, Inc., 2855 Gazelle Court, Carlsbad, CA 92010, USA.
Nucleic Acids Res. 2018 Jan 9;46(1):293-313. doi: 10.1093/nar/gkx1174.
RNase H1-dependent antisense oligonucleotides (ASOs) can degrade complementary RNAs in both the nucleus and the cytoplasm. Since cytoplasmic mRNAs are actively engaged in translation, ASO activity may thus be affected by translating ribosomes that scan the mRNAs. Here we show that mRNAs associated with ribosomes can be cleaved using ASOs and that translation can alter ASO activity. Translation inhibition tends to increase ASO activity when targeting the coding regions of efficiently translated mRNAs, but not nuclear non-coding RNAs or less efficiently translated mRNAs. Increasing the level of RNase H1 protein eliminated the enhancing effects of translation inhibition on ASO activity, suggesting that RNase H1 recruitment to ASO/mRNA heteroduplexes is a rate limiting step and that translating ribosomes can inhibit RNase H1 recruitment. Consistently, ASO activity was not increased by translation inhibition when targeting the 3' UTRs, independent of the translation efficiency of the mRNAs. Contrarily, the activity of 3' UTR-targeting ASOs tended to be reduced upon translation inhibition, likely due to decreased accessibility. These results indicate that ASO activity can be affected by the translation process, and the findings also provide important information toward helping better ASO drug design.
RNase H1 依赖性反义寡核苷酸 (ASO) 可降解核内和细胞质中互补的 RNA。由于细胞质 mRNA 积极参与翻译，因此 ASO 活性可能会受到扫描 mRNA 的翻译核糖体的影响。在这里，我们表明可以使用 ASO 切割与核糖体结合的 mRNA，并且翻译可以改变 ASO 活性。当针对高效翻译的 mRNA 的编码区进行翻译抑制时，翻译抑制往往会增加 ASO 活性，但不会影响核非编码 RNA 或翻译效率较低的 mRNA。增加 RNase H1 蛋白的水平消除了翻译抑制对 ASO 活性的增强作用，这表明 RNase H1 向 ASO/mRNA 异源双链体的募集是限速步骤，并且翻译核糖体可以抑制 RNase H1 的募集。一致地，当针对 3'UTR 进行翻译抑制时，ASO 活性不会增加，而与 mRNA 的翻译效率无关。相反，3'UTR 靶向 ASO 的活性在翻译抑制时趋于降低，可能是由于可及性降低所致。这些结果表明 ASO 活性可能会受到翻译过程的影响，并且这些发现还为更好地设计 ASO 药物提供了重要信息。
