MicroRNAs (miRNAs) provide new therapeutic targets for many diseases, while their myriad roles in development and cellular processes make them fascinating to study. We still do not fully understand the molecular mechanisms by which miRNAs regulate gene expression nor do we know the complete repertoire of mRNAs each miRNA regulates. However, recent progress in the development of effective strategies to block miRNAs suggests that anti-miRNA drugs may soon be used in the clinic.
Understanding the function of individual microRNA (miRNA) species in mice would require the production of hundreds of loss-of-function strains. To accelerate analysis of miRNA biology in mammals, we combined recombinant adeno-associated virus (rAAV) vectors with miRNA `Tough Decoys' (TuDs) to inhibit specific miRNAs. Intravenous injection of rAAV9 expressing anti-miR-122 or anti-let-7 TuD depleted the corresponding miRNA and increased its mRNA targets. rAAV producing anti-miR-122—but not anti-let-7—TuD reduced serum cholesterol by >30% for 25 weeks in wild-type mice. High throughput sequencing of liver miRNAs from the treated mice confirmed that the targeted miRNAs were depleted and revealed that TuD RNAs induce miRNA tailing and trimming in vivo. rAAV-mediated miRNA inhibition thus provides a simple way to study miRNA function in adult mammals and a potential therapy for dyslipidemia and other diseases caused by miRNA deregulation.
Small RNAs loaded into Argonaute proteins direct silencing of complementary target mRNAs. It has been proposed that multiple, imperfectly complementary small interfering RNAs or microRNAs, when bound to the 39 untranslated region of a target mRNA, function cooperatively to silence target expression. We report that, in cultured human HeLa cells and mouse embryonic fibroblasts, Argonaute1 (Ago1), Ago3, and Ago4 act cooperatively to silence both perfectly and partially complementary target RNAs bearing multiple small RNA-binding sites. Our data suggest that for Ago1, Ago3, and Ago4, multiple, adjacent small RNA-binding sites facilitate cooperative interactions that stabilize Argonaute binding. In contrast, small RNAs bound to Ago2 and pairing perfectly to an mRNA target act independently to silence expression. Noncooperative silencing by Ago2 does not require the endoribonuclease activity of the protein: A mutant Ago2 that cannot cleave its mRNA target also silences noncooperatively. We propose that Ago2 binds its targets by a mechanism fundamentally distinct from that used by the three other mammalian Argonaute proteins.
In this issue of Molecular Cell, Denzler et al. (2014) report a quantitative study of microRNA function in adult mouse liver, suggesting that the natural abundance of miRNAs and their binding sites generally excludes the previously proposed regulation of miRNAs by competitive endogenous RNAs (ceRNAs).
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