Hollensen Anne Kruse, Thomsen Rune, Bak Rasmus O, Petersen Charlotte Christie, Ermegaard Eva R, Aagaard Lars, Damgaard Christian Kroun, Mikkelsen Jacob Giehm
Department of Biomedicine, HEALTH, Aarhus University, DK-8000 Aarhus C, Denmark.
Department of Molecular Biology and Genetics, Science and Technology, Aarhus University, DK-8000 Aarhus C, Denmark.
RNA. 2017 Aug;23(8):1247-1258. doi: 10.1261/rna.061192.117. Epub 2017 May 9.
Our genes are post-transcriptionally regulated by microRNAs (miRNAs) inducing translational suppression and degradation of targeted mRNAs. Strategies to inhibit miRNAs in a spatiotemporal manner in a desired cell type or tissue, or at a desired developmental stage, can be crucial for understanding miRNA function and for pushing forward miRNA suppression as a feasible rationale for genetic treatment of disease. For such purposes, RNA polymerase II (RNA Pol II)-transcribed tough decoy (TuD) miRNA inhibitors are particularly attractive. Here, we demonstrate augmented miRNA suppression capacity of TuD RNA hairpins linked to the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). This effect is position-dependent and evident only when the WPRE is positioned upstream of the TuD. In accordance, inclusion of the WPRE does not change nuclear export, translation, total levels of TuD-containing RNA transcripts, or cytoplasmic P-body localization, suggesting that previously reported WPRE functions are negligible for improved TuD function. Notably, deletion analysis of TuD-fused WPRE unveils truncated WPRE variants resulting in optimized miRNA suppression. Together, our findings add to the guidelines for production of WPRE-supported anti-miRNA TuDs.
我们的基因在转录后受到微小RNA（miRNA）的调控，miRNA会诱导靶标mRNA的翻译抑制和降解。在所需细胞类型或组织中，或在所需发育阶段以时空方式抑制miRNA的策略，对于理解miRNA功能以及推动将miRNA抑制作为疾病基因治疗的可行原理至关重要。出于此类目的，由RNA聚合酶II（RNA Pol II）转录的强抑制子（TuD）miRNA抑制剂特别有吸引力。在此，我们证明了与土拨鼠肝炎病毒转录后调控元件（WPRE）相连的TuD RNA发夹增强了miRNA抑制能力。这种效应取决于位置，并且只有当WPRE位于TuD上游时才明显。相应地，WPRE的加入不会改变核输出、翻译、含TuD的RNA转录本的总水平或细胞质P小体定位，这表明先前报道的WPRE功能对于改善TuD功能可忽略不计。值得注意的是，对与TuD融合的WPRE的缺失分析揭示了截短的WPRE变体，其导致了优化的miRNA抑制。总之，我们的发现为生产WPRE支持的抗miRNA TuD提供了指导原则。
