Since their serendipitous discovery in nematodes, microRNAs (miRNAs) have emerged as key regulators of biological processes in animals. These small RNAs form complex networks that regulate cell differentiation, development and homeostasis. Deregulation of miRNA function is associated with an increasing number of human diseases, particularly cancer. Recent discoveries have expanded our understanding of the control of miRNA function. Here, we review the mechanisms that modulate miRNA activity, stability and cellular localization through alternative processing and maturation, sequence editing, post-translational modifications of Argonaute proteins, viral factors, transport from the cytoplasm and regulation of miRNA-target interactions. We conclude by discussing intriguing, unresolved research questions.

The structural flexibility of RNA underlies fundamental biological processes, but there are no methods for exploring the multiple conformations adopted by RNAs in vivo. We developed cross-linking of matched RNAs and deep sequencing (COMRADES) for in-depth RNA conformation capture, and a pipeline for the retrieval of RNA structural ensembles. Using COMRADES, we determined the architecture of the Zika virus RNA genome inside cells, and identified multiple site-specific interactions with human noncoding RNAs.

microRNAs (miRNAs) guide Argonaute proteins to mRNAs targeted for repression. Target recognition occurs primarily through the miRNA seed region, composed of guide (g) nucleotides g2-g8. However, nucleotides beyond the seed are also important for some known miRNA-target interactions. Here, we report the structure of human Argonaute2 (Ago2) engaged with a target RNA recognized through both miRNA seed and supplementary (g13-g16) regions. Ago2 creates a "supplementary chamber" that accommodates up to five miRNA-target base pairs. Seed and supplementary chambers are adjacent to each other and can be bridged by an unstructured target loop of 1-15 nucleotides. Opening of the supplementary chamber may be constrained by tension in the miRNA 3 0 tail, as increases in miRNA length stabilize supplementary interactions. Contrary to previous reports, we demonstrate that optimal supplementary interactions can increase target affinity > 20-fold. These results provide a mechanism for extended miRNA targeting, suggest a function for 3 0 isomiRs in tuning miRNA targeting specificity, and indicate that supplementary interactions may contribute more to target recognition than is widely appreciated.

MicroRNAs (miRNAs) are short noncoding RNAs, which bind to messenger RNAs and regulate protein expression. The biosynthesis of miRNAs includes two precursors, a primary miRNA transcript (pri-miRNA) and a shorter pre-miRNA, both of which carry a common stem-loop bearing the mature miRNA. MiR-122 is a liver-specific miRNA with an important role in the life cycle of hepatitis C virus (HCV). It is the target of miravirsen (SPC3649), an antimiR drug candidate currently in clinical testing for treatment of HCV infections. Miravirsen is composed of locked nucleic acid (LNAs) ribonucleotides interspaced throughout a DNA phosphorothioate sequence complementary to mature miR-122. The LNA modifications endow the drug with high affinity for its target and provide resistance to nuclease degradation. While miravirsen is thought to work mainly by hybridizing to mature miR-122 and blocking its interaction with HCV RNA, its target sequence is also present in pri- and pre-miR-122. Using new in vitro and cellular assays specifically developed to discover ligands that suppress biogenesis of miR-122, we show that miravirsen binds to the stem-loop structure of pri- and pre-miR-122 with nanomolar affinity, and inhibits both Dicer- and Drosha-mediated processing of miR-122 precursors. This inhibition may contribute to the pharmacological activity of the drug in man.

Lin28 inhibits the biogenesis of let-7 miRNAs through a direct interaction with the terminal loop of pre-let-7. This interaction requires the zinc-knuckle domains of Lin28. We show that the zinc knuckle domains of Lin28 are sufficient to provide binding selectivity for pre-let-7 miRNAs and present the NMR structure of human Lin28 zinc knuckles bound to the short sequence 5'-AGGAGAU-3'. The structure reveals that each zinc knuckle recognizes an AG dinucleotide separated by a single nucleotide spacer. This defines a new 5'-NGNNG-3' consensus motif that explains how Lin28 selectively recognizes pre-let-7 family members. Binding assays in cell lysates and functional assays in cultured cells demonstrate that the interactions observed in the solution structure also occur between the full-length protein and members of the pre-let-7 family. The consensus sequence explains several seemingly disparate previously published observations on the binding properties of Lin28.