Division of Genomics and RNomics, Innsbruck Biocentre, Innsbruck Medical University, Innsbruck and Institute of Theoretical Chemistry, University of Vienna, Vienna, Austria.
Nucleic Acids Res. 2010 Jun;38(10):e113. doi: 10.1093/nar/gkq057. Epub 2010 Feb 11.
Up to 450,000 non-coding RNAs (ncRNAs) have been predicted to be transcribed from the human genome. However, it still has to be elucidated which of these transcripts represent functional ncRNAs. Since all functional ncRNAs in Eukarya form ribonucleo-protein particles (RNPs), we generated specialized cDNA libraries from size-fractionated RNPs and validated the presence of selected ncRNAs within RNPs by glycerol gradient centrifugation. As a proof of concept, we applied the RNP method to human Hela cells or total mouse brain, and subjected cDNA libraries, generated from the two model systems, to deep-sequencing. Bioinformatical analysis of cDNA sequences revealed several hundred ncRNP candidates. Thereby, ncRNAs candidates were mainly located in intergenic as well as intronic regions of the genome, with a significant overrepresentation of intron-derived ncRNA sequences. Additionally, a number of ncRNAs mapped to repetitive sequences. Thus, our RNP approach provides an efficient way to identify new functional small ncRNA candidates, involved in RNP formation.
据预测，人类基因组可转录多达 45 万个非编码 RNA（ncRNA）。然而，这些转录本中哪些代表功能性 ncRNA 仍有待阐明。由于真核生物中所有功能性 ncRNA 都形成核糖核蛋白颗粒（RNP），我们从大小分级的 RNP 中生成了专门的 cDNA 文库，并通过甘油梯度离心验证了选定的 ncRNA 在 RNP 中的存在。作为概念验证，我们将 RNP 方法应用于人类 Hela 细胞或总鼠脑，并对来自两个模型系统的 cDNA 文库进行深度测序。对 cDNA 序列的生物信息学分析揭示了数百个 ncRNP 候选物。因此，ncRNA 候选物主要位于基因组的基因间区和内含子区，内含子衍生的 ncRNA 序列显著过表达。此外，一些 ncRNA 映射到重复序列。因此，我们的 RNP 方法提供了一种有效的方法来识别新的功能性小 ncRNA 候选物，这些候选物参与 RNP 的形成。
