title: Synthetic pre-microRNAs reveal dual-strand activity of miR-34a on TNF-α.
abstract: Functional microRNAs (miRNAs) are produced from both arms of their precursors (pre-miRNAs). Their abundances vary in context-dependent fashion spatiotemporarily and there is mounting evidence of regulatory interplay between them. Here, we introduce chemically synthesized pre-miRNAs (syn-pre-miRNAs) as a general class of accessible, easily transfectable mimics of pre-miRNAs. These are RNA hairpins, identical in sequence to natural pre-miRNAs. They differ from commercially available miRNA mimics through their complete hairpin structure, including any regulatory elements in their terminal-loop regions and their potential to introduce both strands into RISC. They are distinguished from transcribed pre-miRNAs by their terminal 5' hydroxyl groups and their precisely defined terminal nucleotides. We demonstrate with several examples how they fully recapitulate the properties of pre-miRNAs, including their processing by Dicer into functionally active 5p; and 3p-derived mature miRNAs. We use syn-pre-miRNAs to show that miR-34a uses its 5p and 3p miRNAs in two pathways: apoptosis during TGF-β signaling, where SIRT1 and SP4 are suppressed by miR-34a-5p and miR-34a-3p, respectively; and the lipopolysaccharide (LPS)-activation of primary human monocyte-derived macrophages, where TNF (TNFα) is suppressed by miR-34a-5p indirectly and miR-34a-3p directly. Our results add to growing evidence that the use of both arms of a miRNA may be a widely used mechanism. We further suggest that syn-pre-miRNAs are ideal and affordable tools to investigate these mechanisms.
authors.forename: Boris
authors.initials: B
authors.lastname: Guennewig
authors.forename: Martina
authors.initials: M
authors.lastname: Roos
authors.forename: Afzal M
authors.initials: AM
authors.lastname: Dogar
authors.forename: Luca F R
authors.initials: LF
authors.lastname: Gebert
authors.forename: Julian A
authors.initials: JA
authors.lastname: Zagalak
authors.forename: Valentina
authors.initials: V
authors.lastname: Vongrad
authors.forename: Karin J
authors.initials: KJ
authors.lastname: Metzner
authors.forename: Jonathan
authors.initials: J
authors.lastname: Hall
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D035683:MicroRNAs / Q000138:chemical synthesis / Q000502:physiology*; D012322:RNA Precursors / Q000138:chemical synthesis / Q000502:physiology; D012330:RNA, Double-Stranded / Q000138:chemical synthesis / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
chemical_list: D008070:Lipopolysaccharides; C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D012322:RNA Precursors; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha
keywords: 3p strand; TNF-α; macrophages; miR-34a; miRNA biogenesis; pre-miRNA