Contrant Maud, Fender Aurélie, Chane-Woon-Ming Béatrice, Randrianjafy Ramy, Vivet-Boudou Valérie, Richer Delphine, Pfeffer Sébastien
Architecture et Réactivité de l'ARN - UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, 15 rue René Descartes, F-67084 Strasbourg Cedex, France.
Architecture et Réactivité de l'ARN - UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, 15 rue René Descartes, F-67084 Strasbourg Cedex, France
Nucleic Acids Res. 2014 Jul;42(12):7981-96. doi: 10.1093/nar/gku424. Epub 2014 May 15.
Micro (mi)RNAs are small non-coding RNAs with key regulatory functions. Recent advances in the field allowed researchers to identify their targets. However, much less is known regarding the regulation of miRNAs themselves. The accumulation of these tiny regulators can be modulated at various levels during their biogenesis from the transcription of the primary transcript (pri-miRNA) to the stability of the mature miRNA. Here, we studied the importance of the pri-miRNA secondary structure for the regulation of mature miRNA accumulation. To this end, we used the Kaposi's sarcoma herpesvirus, which encodes a cluster of 12 pre-miRNAs. Using small RNA profiling and quantitative northern blot analysis, we measured the absolute amount of each mature miRNAs in different cellular context. We found that the difference in expression between the least and most expressed viral miRNAs could be as high as 60-fold. Using high-throughput selective 2'-hydroxyl acylation analyzed by primer extension, we then determined the secondary structure of the long primary transcript. We found that highly expressed miRNAs derived from optimally structured regions within the pri-miRNA. Finally, we confirmed the importance of the local structure by swapping stem-loops or by targeted mutagenesis of selected miRNAs, which resulted in a perturbed accumulation of the mature miRNA.
微小（mi）RNA是具有关键调控功能的小型非编码RNA。该领域的最新进展使研究人员能够识别其靶标。然而，关于miRNA自身的调控却知之甚少。这些微小调控因子的积累在其从初级转录本（pri-miRNA）转录到成熟miRNA稳定性的生物合成过程中的各个水平上都可受到调节。在此，我们研究了pri-miRNA二级结构对成熟miRNA积累调控的重要性。为此，我们使用了卡波西肉瘤疱疹病毒，其编码一组12个pre-miRNA。通过小RNA谱分析和定量Northern印迹分析，我们测量了不同细胞环境中每种成熟miRNA的绝对量。我们发现，表达最少和最多的病毒miRNA之间的表达差异可能高达60倍。然后，通过引物延伸高通量选择性2'-羟基酰化分析，我们确定了长初级转录本的二级结构。我们发现，高表达的miRNA源自pri-miRNA内结构最佳的区域。最后，我们通过交换茎环或对选定的miRNA进行靶向诱变来证实局部结构的重要性，这导致成熟miRNA的积累受到干扰。
