Liu Ying Poi, Karg Margarete, Harwig Alex, Herrera-Carrillo Elena, Jongejan Aldo, van Kampen Antoine, Berkhout Ben
a Laboratory of Experimental Virology; Department of Medical Microbiology; Center for Infection and Immunity Amsterdam (CINIMA); Academic Medical Center; University of Amsterdam ; Amsterdam , The Netherlands.
RNA Biol. 2015;12(1):92-100. doi: 10.1080/15476286.2015.1017204.
Short hairpin RNAs (shRNAs) are widely used for gene knockdown by inducing the RNA interference (RNAi) mechanism, both for research and therapeutic purposes. The shRNA precursor is processed by the RNase III-like enzyme Dicer into biologically active small interfering RNA (siRNA). This effector molecule subsequently targets a complementary mRNA for destruction via the Argonaute 2 (AGO2) complex. The cellular role of Dicer concerns the processing of pre-miRNAs into mature microRNA (miRNA). Recently, a non-canonical pathway was reported for the biogenesis of miR-451, which bypasses Dicer and is processed instead by the slicer activity of AGO2, followed by the regular AGO2-mediated mRNA targeting step. Interestingly, shRNA designs that are characterized by a relatively short basepaired stem also bypass Dicer to be processed by AGO2. We named this design AgoshRNA as these molecules depend on AGO2 both for processing and silencing activity. In this study, we investigated diverse mechanistic aspects of this new class of AgoshRNA molecules. We probed the requirements for AGO2-mediated processing of AgoshRNAs by modification of the proposed cleavage site in the hairpin. We demonstrate by deep sequencing that AGO2-processed AgoshRNAs produce RNA effector molecules with more discrete ends than the products of the regular shRNA design. Furthermore, we tested whether trimming and tailing occurs upon AGO2-mediated processing of AgoshRNAs, similar to what has been described for miR-451. Finally, we tested the prediction that AgoshRNA activity, unlike that of regular shRNAs, is maintained in Dicer-deficient cell types. These mechanistic insights could aid in the design of optimised AgoshRNA tools and therapeutics.
短发夹RNA（shRNA）通过诱导RNA干扰（RNAi）机制被广泛用于基因敲低，用于研究和治疗目的。shRNA前体由核糖核酸酶III样酶Dicer加工成具有生物活性的小干扰RNA（siRNA）。这种效应分子随后靶向互补mRNA，通过Argonaute 2（AGO2）复合物将其破坏。Dicer在细胞中的作用涉及将前体miRNA加工成成熟的微小RNA（miRNA）。最近，报道了miR-451生物合成的一种非经典途径，该途径绕过Dicer，而是由AGO2的切割活性进行加工，随后是常规的AGO2介导的mRNA靶向步骤。有趣的是，以相对较短的碱基配对茎为特征的shRNA设计也绕过Dicer，由AGO2进行加工。我们将这种设计命名为AgoshRNA，因为这些分子的加工和沉默活性都依赖于AGO2。在本研究中，我们研究了这类新型AgoshRNA分子的各种机制方面。我们通过修饰发夹中提议的切割位点来探究AGO2介导的AgoshRNAs加工的要求。我们通过深度测序证明，AGO2加工的AgoshRNAs产生的RNA效应分子的末端比常规shRNA设计的产物更离散。此外，我们测试了在AGO2介导的AgoshRNAs加工过程中是否会发生修剪和加尾，这与miR-451的情况类似。最后，我们测试了一个预测，即与常规shRNAs不同，AgoshRNA活性在Dicer缺陷细胞类型中得以维持。这些机制上的见解有助于优化AgoshRNA工具和治疗方法的设计。
