Methodological obstacles in knocking down small noncoding RNAs
Methodological obstacles in knocking down small noncoding RNAs
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DOI：
10.1261/rna.1740009
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发表时间：
2009-10-01
期刊：
影响因子：
4.5
通讯作者：
Huettenhofer, Alexander
中科院分区：
文献类型：
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作者：
Ploner, Andreas;Ploner, Christian;Huettenhofer, Alexander
在最近的过去，几千个非编码RNA （ncRNA）基因在真核基因组中被预测。然而，对于它们的功能分析，目前只有少数高通量方法可用于敲除选定的ncRNA物种，例如被称为antagomirs的反义探针靶向的microrna。因此，我们比较了四种敲低策略的效率，这些策略以前主要用于分析蛋白质编码基因，以研究ncrna的功能，特别是小核核rna （snoRNAs）。因此，该类snorna代表了最丰富的ncRNA物种之一。大多数snoRNAs已被证明通过互补反义元件靶向核糖体rna （RNAs）介导核苷酸修饰。然而，一些被称为“孤儿snorna”的snorna与rnas缺乏明显的互补性，因此它们的功能仍然难以捉摸。因此，我们应用RNA干扰（RNAi）、锁定核酸（LNA）或肽核酸反义方法，以及基于核酶的策略来敲除snoRNA。作为原理证明，我们瞄准了典型的U81 snoRNA，它已被证明可以介导真核28S rRNA中核苷酸a（391）的修饰。我们的研究结果表明，虽然RNAi是一个不适合的工具来敲除snoRNA，但基于核酶的策略，以及lna -反义寡核苷酸方法，导致U81 snoRNA表达水平降低高达60%。然而，没有观察到U81 snoRNA的酶活性随之下降，这表明未来需要改进更有效的ncrna敲低技术。
In the recent past, several thousand noncoding RNA (ncRNA) genes have been predicted within eukaryal genomes. However, for their functional analysis only a few high-throughput methods are currently available to knock down selected ncRNA species, such as microRNAs, which are targeted by antisense probes, termed antagomirs. We thus compared the efficiencies of four knockdown strategies, previously mainly employed for the analysis of protein-coding genes, to study the function of ncRNAs, in particular, small nucleolar RNAs (snoRNAs). Thereby, the class of snoRNAs represents one of the most abundant ncRNA species. The majority of snoRNAs has been shown to mediate nucleotide modifications by targeting ribosomal RNAs (rRNAs) through complementary antisense elements. However, some snoRNAs, termed "orphan snoRNAs," lack telltale complementarities to rRNAs and thus their function remains elusive. We therefore applied RNA interference (RNAi), locked nucleic acid (LNA), or peptide nucleic acid antisense approaches, as well as a ribozyme-based strategy to knock down a snoRNA. As a proof of principle, we targeted the canonical U81 snoRNA, which has been shown to mediate modification of nucleotide A(391) within eukaryal 28S rRNA. Our results demonstrate that while RNAi is an unsuitable tool for snoRNA knockdown, a ribozyme-based strategy, as well as an LNA-antisense oligonucleotide approach, resulted in a decrease of U81 snoRNA expression levels up to 60%. However, no concomitant decrease in enzymatic activity of U81 snoRNA was observed, indicating that improvement of more efficient knockdown techniques for ncRNAs will be required in the future.