Mechanistic insights on the Dicer-independent AGO2-mediated processing of AgoshRNAs

摘要: Short hairpin RNAs (shRNAs) are widely used for gene knockdown by inducing the RNA interference (RNAi) mechanism, both research and therapeutic purposes. The shRNA precursor is processed RNase III-like enzyme Dicer into biologically active small interfering (siRNA). This effector molecule subsequently targets a complementary mRNA destruction via Argonaute 2 (AGO2) complex. cellular role of concerns processing pre-miRNAs mature microRNA (miRNA). Recently, non-canonical pathway was reported biogenesis miR-451, which bypasses instead slicer activity AGO2, followed regular AGO2-mediated targeting step. Interestingly, designs that characterized relatively short basepaired stem also bypass to be AGO2. We named this design AgoshRNA as these molecules depend on AGO2 silencing activity. In study, we investigated diverse mechanistic aspects new class molecules. probed requirements AgoshRNAs modification proposed cleavage site in hairpin. demonstrate deep sequencing AGO2-processed produce with more discrete ends than products design. Furthermore, tested whether trimming tailing occurs upon AgoshRNAs, similar what has been described miR-451. Finally, prediction activity, unlike shRNAs, maintained Dicer-deficient cell types. These insights could aid optimised tools therapeutics.