Synthetic pre-microRNAs reveal dual-strand activity of miR-34a on TNF-[alpha]

• Published in: RNA (Cambridge) 2014-01, Vol.20 (1), p.61-75
• Main Authors: Guennewig, Boris, Roos, Martina, Dogar, Afzal M, Gebert, Luca Fr, ZAGALAK, JULIAN A, VONGRAD, VALENTINA, Metzner, Karin J, Hall, Jonathan
• Format: Article
• Language: English
• ISSN: 1355-8382
• DOI: 10.1261/rna.038968.113

Functional microRN As (miRNAs) are produced from both arms of their precursors (pre-miRN As). Their abundances vary in contextdependent 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 premiRNAs. 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-[beta] 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 (TNF[alpha]) 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.