Regulation of microRNA function in animals.

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.

Assembly of the bacterial ribosome with circularly permuted rRNA.

Co-transcriptional assembly is an integral feature of the formation of RNA-protein complexes that mediate translation. For ribosome synthesis, prior studies have indicated that the strict order of transcription of rRNA domains may not be obligatory during bacterial ribosome biogenesis, since a series of circularly permuted rRNAs are viable. In this work, we report the structural insights into assembly of the bacterial ribosome large subunit (LSU) based on cryo-EM density maps of intermediates that accumulate during in vitro ribosome synthesis using a set of circularly permuted (CiPer) rRNAs. The observed ensemble of 23 resolved ribosome large subunit intermediates reveals conserved assembly routes with an underlying hierarchy among cooperative assembly blocks. There are intricate interdependencies for the formation of key structural rRNA helices revealed from the circular permutation of rRNA. While the order of domain synthesis is not obligatory, the order of domain association does appear to proceed with a particular order, likely due to the strong evolutionary pressure on efficient ribosome synthesis. This work reinforces the robustness of the known assembly hierarchy of the bacterial large ribosomal subunit and offers a coherent view of how efficient assembly of CiPer rRNAs can be understood in that context.

miR-122-based therapies select for three distinct resistance mechanisms based on alterations in RNA structure.

Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with a liver-specific microRNA called miR-122. miR-122 binds to two sites in the 5' untranslated region of the viral genome and promotes HCV RNA accumulation. This interaction is important for viral RNA accumulation in cell culture, and miR-122 inhibitors have been shown to be effective at reducing viral titers in chronic HCV-infected patients. Herein, we analyzed resistance-associated variants that were isolated in cell culture or from patients who underwent miR-122 inhibitor-based therapy and discovered three distinct resistance mechanisms all based on changes to the structure of the viral RNA. Specifically, resistance-associated variants promoted riboswitch activity, genome stability, or positive-strand viral RNA synthesis, all in the absence of miR-122. Taken together, these findings provide insight into the mechanism(s) of miR-122-mediated viral RNA accumulation and provide mechanisms of antiviral resistance mediated by changes in RNA structure.

MiR-CLIP reveals iso-miR selective regulation in the miR-124 targetome.

Many microRNAs regulate gene expression via atypical mechanisms, which are difficult to discern using native cross-linking methods. To ascertain the scope of non-canonical miRNA targeting, methods are needed that identify all targets of a given miRNA. We designed a new class of miR-CLIP probe, whereby psoralen is conjugated to the 3p arm of a pre-microRNA to capture targetomes of miR-124 and miR-132 in HEK293T cells. Processing of pre-miR-124 yields miR-124 and a 5'-extended isoform, iso-miR-124. Using miR-CLIP, we identified overlapping targetomes from both isoforms. From a set of 16 targets, 13 were differently inhibited at mRNA/protein levels by the isoforms. Moreover, delivery of pre-miR-124 into cells repressed these targets more strongly than individual treatments with miR-124 and iso-miR-124, suggesting that isomirs from one pre-miRNA may function synergistically. By mining the miR-CLIP targetome, we identified nine G-bulged target-sites that are regulated at the protein level by miR-124 but not isomiR-124. Using structural data, we propose a model involving AGO2 helix-7 that suggests why only miR-124 can engage these sites. In summary, access to the miR-124 targetome via miR-CLIP revealed for the first time how heterogeneous processing of miRNAs combined with non-canonical targeting mechanisms expand the regulatory range of a miRNA.

COMRADES determines in vivo RNA structures and interactions.

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.

miR-122 and Ago interactions with the HCV genome alter the structure of the viral 5' terminus.

Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with the liver-specific microRNA, miR-122. miR-122 binds to two sites in the 5' untranslated region (UTR) and this interaction promotes HCV RNA accumulation, although the precise role of miR-122 in the HCV life cycle remains unclear. Using biophysical analyses and Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE) we investigated miR-122 interactions with the 5' UTR. Our data suggests that miR-122 binding results in alteration of nucleotides 1-117 to suppress an alternative secondary structure and promote functional internal ribosomal entry site (IRES) formation. Furthermore, we demonstrate that two hAgo2:miR-122 complexes are able to bind to the HCV 5' terminus simultaneously and SHAPE analyses revealed further alterations to the structure of the 5' UTR to accommodate these complexes. Finally, we present a computational model of the hAgo2:miR-122:HCV RNA complex at the 5' terminus of the viral genome as well as hAgo2:miR-122 interactions with the IRES-40S complex that suggest hAgo2 is likely to form additional interactions with SLII which may further stabilize the HCV IRES. Taken together, our results support a model whereby hAgo2:miR-122 complexes alter the structure of the viral 5' terminus and promote formation of the HCV IRES.

Synthetic pre-microRNAs reveal dual-strand activity of miR-34a on TNF-α.

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-ß 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.

Miravirsen (SPC3649) can inhibit the biogenesis of miR-122.

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.

Assembly of the Bacterial Ribosome with Circularly Permuted rRNA.

Co-transcriptional assembly is an integral feature of the formation of RNA-protein complexes that mediate translation. For ribosome synthesis, prior studies have indicated that the strict order of transcription of rRNA domains may not be obligatory during bacterial ribosome biogenesis, since a series of circularly permuted rRNAs are viable. In this work, we report the insights into assembly of the bacterial ribosome large subunit (LSU) based on cryo-EM density maps of intermediates that accumulate during in vitro ribosome synthesis using a set of circularly permuted (CiPer) rRNAs. The observed ensemble of twenty-three resolved ribosome large subunit intermediates reveals conserved assembly routes with an underlying hierarchy among cooperative assembly blocks. There are intricate interdependencies for the formation of key structural rRNA helices revealed from the circular permutation of rRNA. While the order of domain synthesis is not obligatory, the order of domain association does appear to proceed with a particular order, likely due to the strong evolutionary pressure on efficient ribosome synthesis. This work reinforces the robustness of the known assembly hierarchy of the bacterial large ribosomal subunit, and offers a coherent view of how efficient assembly of CiPer rRNAs can be understood in that context.

A structured RNA motif locks Argonaute2:miR-122 onto the 5' end of the HCV genome.

microRNAs (miRNAs) form regulatory networks in metazoans. Viruses engage miRNA networks in numerous ways, with Flaviviridae members exploiting direct interactions of their RNA genomes with host miRNAs. For hepatitis C virus (HCV), binding of liver-abundant miR-122 stabilizes the viral RNA and regulates viral translation. Here, we investigate the structural basis for these activities, taking into consideration that miRNAs function in complex with Argonaute (Ago) proteins. The crystal structure of the Ago2:miR-122:HCV complex reveals a structured RNA motif that traps Ago2 on the viral RNA, masking its 5' end from enzymatic attack. The trapped Ago2 can recruit host factor PCBP2, implicated in viral translation, while binding of a second Ago2:miR-122 competes with PCBP2, creating a potential molecular switch for translational control. Combined results reveal a viral RNA structure that modulates Ago2:miR-122 dynamics and repurposes host proteins to generate a functional analog of the mRNA cap-binding complex.

Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28.

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.