Interleukin-6 mRNA is unstable and degraded with a half-life of 30 min. Instability determinants can entirely be attributed to the 3 untranslated region. By grafting segments of this region to stable green fluorescent protein mRNA and subsequent scanning mutagenesis, we have identified two conserved elements, which together account for most of the instability. The first corresponds to a short noncanonical AU-rich element. The other, 80 nucleotides further 5, comprises a sequence predicted to form a stem-loop structure. Neither element alone was sufficient to confer full instability, suggesting that they might cooperate. Overexpression of myc-tagged AUF1 p37 and p42 isoforms as well as suppression of endogenous AUF1 by RNA interference stabilized interleukin-6 mRNA. Both effects required the AU-rich instability element. Similarly, the proteasome inhibitor MG132 stabilized interleukin-6 mRNA probably through an increase of AUF1 levels. The mRNA coimmunoprecipitated specifically with myc-tagged AUF1 p37 and p42 in cell extracts but only when the AU-rich instability element was present. These results indicate that AUF1 binds to the AU-rich element in vivo and promotes IL-6 mRNA degradation.

Functional microRNAs (miRNAs) are produced from both arms of their precursors (pre-miRNAs). 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-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.

No abstract

Cancer cells secreting excess latent TGF-β are often resistant to TGF-β induced growth inhibition. We observed that RNAi against TGF-β1 led to apoptotic death in such cell lines with features that were, paradoxically, reminiscent of TGF-β signaling activity and that included transiently enhanced SMAD2 and AKT phosphorylation. A comprehensive search in Hela cells for potential microRNA drivers of this mechanism revealed that RNAi against TGF-β1 led to induction of pro-apoptotic miR-34a and to a globally decreased oncomir expression. The reduced levels of the oncomirs miR-18a and miR-24 accounted for the observed derepression of two TGF-β1 processing factors, thrombospondin-1, and furin, respectively. Our data suggest a novel mechanism in which latent TGF-β1, thrombospondin 1, and furin form a microRNA-mediated regulatory feedback loop. For cells with high levels of latent TGF-β, this provides a potentially widespread mechanism of escape from TGF-β-mediated growth arrest at the earliest point in the signaling pathway, TGF-β processing.

Experiments conducted with micro RNA (miRNA) mimics often result in subtle phenotypic changes and hence require careful controls. A commonly used type of control reagent in the antisense/RNA interference fields is the mismatched sequence. However, it is difficult to use mismatch controls for miRNAs, mainly because base permutation in the seed region may generate a new miRNA seed with its own associated target transcripts. We incorporated N(4)-methylcytidine and N(4),N(4)-dimethylcytidine into a series of RNAs using the convertible nucleoside approach and measured their effects on hybridization affinity with complementary RNAs, and on miRNA-mediated and small interfering RNA (SiRNA)-mediated silencing. We report here that incorporation of a single N(4),N(4)-dimethylcytidine into the seed region of miRNAs can be used as a new class of negative miRNA control which (1) does not constitute a new seed sequence; (2) is accepted by the RNA-induced silencing complex (RISC); (3) causes a significant loss of binding affinity to target RNAs; and (4) is synthesized conveniently into oligoribonucleotides.