When Argonaute takes out the ribonuclease sword.

Argonaute (AGO) proteins in all three domains of life form ribonucleoprotein or deoxyribonucleoprotein complexes by loading a guide RNA or DNA, respectively. Since all AGOs retain a PIWI domain that takes an RNase H fold, the ancestor was likely an endoribonuclease (i.e., a slicer). In animals, most miRNA-mediated gene silencing occurs slicer independently. However, the slicer activity of AGO is indispensable in specific events, such as development and differentiation, which are critical for vertebrates and thus cannot be replaced by the slicer-independent regulation. This review highlights the distinctions in catalytic activation mechanisms among slicing-competent AGOs, shedding light on the roles of two metal ions in target recognition and cleavage. The precision of the target specificity by the RNA-induced silencing complexes is reevaluated and redefined. The possible coevolutionary relationship between slicer-independent gene regulation and AGO-binding protein, GW182, is also explored. These discussions reveal that numerous captivating questions remain unanswered regarding the timing and manner in which AGOs employ their slicing activity.

The Multiplicity of Argonaute Complexes in Mammalian Cells.

Argonautes (AGOs) are a highly conserved family of proteins found in most eukaryotes and involved in mechanisms of gene regulation, both at the transcriptional and post-transcriptional level. Among other functions, AGO proteins associate with microRNAs (miRNAs) to mediate the post-transcriptional repression of protein-coding genes. In this process, AGOs associate with members of the trinucleotide repeat containing 6 protein (TNRC6) family to form the core of the RNA-induced silencing complex (RISC), the effector machinery that mediates miRNA function. However, the description of the exact composition of the RISC has been a challenging task due to the fact the AGO's interactome is dynamically regulated in a cell type- and condition-specific manner. Here, we summarize some of the most significant studies that have identified AGO complexes in mammalian cells, as well as the approaches used to characterize them. Finally, we discuss possible opportunities to exploit what we have learned on the properties of the RISC to develop novel anti-cancer therapies. SIGNIFICANCE STATEMENT: The RNA-induced silencing complex (RISC) is the molecular machinery that mediates miRNA function in mammals. Studies over the past two decades have shed light on important biochemical and functional properties of this complex. However, many aspects of this complex await further elucidation, mostly due to technical limitations that have hindered full characterization. Here, we summarize some of the most significant studies on the mammalian RISC and discuss possible sources of biases in the approaches used to characterize it.

Roles for Drosophila cap1 2'-O-ribose methyltransferase in the small RNA silencing pathway associated with Argonaute 2.

Cap1 2'-O-ribose methyltransferase (CMTR1) modifies RNA transcripts containing the 7-methylguanosine cap via 2'-O-ribose methylation of the first transcribed nucleotide, yielding cap1 structures. However, the role of CMTR1 in small RNA-mediated gene silencing remains unknown. Here, we identified and characterized a Drosophila CMTR1 gene (dCMTR1) mutation. We found that the catalytic activity of dCMTR1 was involved in the biogenesis of small interfering RNAs (siRNAs) but not microRNAs. Additionally, dCMTR1 interacted with R2D2, a key component for the assembly of the RNA-induced silencing complex (RISC) containing Argonaute 2 (Ago2). Consistent with this finding, loss of dCMTR1 function impaired RISC assembly by inhibiting the unwinding of Ago2-bound siRNA duplexes, thus preventing the retention of the guide strand. Moreover, dCMTR1 is unlikely to modify siRNAs during RISC assembly. Collectively, our data indicate that dCMTR1 is a positive regulator of the small RNA pathway associated with Ago2 with roles in both siRNA biogenesis and RISC assembly.

A universal method for the rapid isolation of all known classes of functional silencing small RNAs.

Diverse classes of silencing small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Here, we have streamlined various embodiments of a Q-sepharose-based RISC-purification method that relies on conserved biochemical properties of all ARGONAUTEs. We show, in multiple benchmarking assays, that the resulting 15-min benchtop extraction procedure allows simultaneous purification of all known classes of RISC-associated sRNAs without prior knowledge of the samples-intrinsic ARGONAUTE repertoires. Optimized under a user-friendly format, the method - coined 'TraPR' for Trans-kingdom, rapid, affordable Purification of RISCs - operates irrespectively of the organism, tissue, cell type or bio-fluid of interest, and scales to minute amounts of input material. The method is highly suited for direct profiling of silencing sRNAs, with TraPR-generated sequencing libraries outperforming those obtained via gold-standard procedures that require immunoprecipitations and/or lengthy polyacrylamide gel-selection. TraPR considerably improves the quality and consistency of silencing sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots or mammalian plasma, and regardless of RNA contaminants or RNA degradation status of samples.

Redirection of miRNA-Argonaute Complexes to Specific Target Sites by Synthetic Adaptor Molecules.

Dysregulation of miRNAs is connected with a multitude of diseases for which antagomirs and miRNA replacement are discussed as therapeutic options. Here, we suggest an alternative concept based on the redirection of RISCs to non-native target sites. Metabolically stable DNA-LNA mixmers are used to mediate the binding of RISCs to mRNAs without any direct base complementarity to the presented guide RNA strand. Physical redirection of a dye-labeled miRNA model and of specific miRNA-programmed RISC fractions present in HeLa extracts is demonstrated by pull-down experiments with biotinylated capture oligonucleotides.

Monitoring Dicer-Mediated miRNA-21 Maturation and Ago2 Loading by a Dual-Colour FIT PNA Probe Set.

The inhibition of micro RNA (miRNA) maturation by Dicer and loading matured miRNAs into the RNA-induced silencing complex (RISC) is envisioned as a modality for treatment of cancer. Existing methods for evaluating maturation either focus on the conversion of modified precursors or detect mature miRNA. Whereas the former is not applicable to native pre-miRNA, the latter approach underestimates maturation when both nonmatured and matured miRNA molecules are subject to cleavage. We present a set of two orthogonally labelled FIT PNA probes that distinguish between cleaved pre-miRNA and the mature miRNA duplex. The probes allow Dicer-mediated miR21 maturation to be monitored and Ago2-mediated unwinding of the miR21 duplex to be assayed. A two-channel fluorescence readout enables measurement in real-time without the need for specialized instrumentation or further enzyme mediated amplification.

Structural improvement of LidNA: delta-type LidNA is a potent miRNA inhibitor constructed with unmodified DNA.

Many miRNA inhibitors have been developed, including chemically modified oligonucleotides, such as 2'-O-methylated RNA and locked nucleic acid (LNA). Unmodified DNA has not yet been reported as a miRNA inhibitor due to relatively low DNA/miRNA binding affinity. We designed a structured DNA, LidNA, which was constructed with unmodified DNA, consisting of a complementary sequence to the target miRNA flanked by two structured DNA regions, such as double-stranded DNA. LidNA inhibited miRNA activity more potently than 2'-O-methylated RNA or LNA. To optimize LidNA, two double-stranded regions were joined, causing the molecule to assume a delta-like shape, which we termed delta-type LidNA. Delta-type LidNAs were developed to target endogenous and exogenous miRNAs, and exhibited potent miRNA inhibitory effects with a duration of at least 10 days. Delta-type LidNA-21, which targeted miR-21, inhibited the growth of cancer cell lines. This newly developed LidNA could contribute to miRNA studies across multiple fields.Abbreviations: LidNA: DNA that puts a lid on miRNA function; LNA: locked nucleic acid; 3'-UTR: 3'-untranslated regions; RISC: RNA-induced silencing complex; MBL: Molecular beacon-like LidNA; YMBL: Y-type molecular beacon-like LidNA; TDMD: target-directed microRNA degradation.

Multidomain Convergence of Argonaute during RISC Assembly Correlates with the Formation of Internal Water Clusters.

Despite the relevance of Argonaute proteins in RNA silencing, little is known about the structural steps of small RNA loading to form RNA-induced silencing complexes (RISCs). We report the 1.9 Å crystal structure of human Argonaute4 with guide RNA. Comparison with the previously determined apo structure of Neurospora crassa QDE2 revealed that the PIWI domain has two subdomains. Binding of guide RNA fastens the subdomains, thereby rearranging the active-site residues and increasing the affinity for TNRC6 proteins. We also identified two water pockets beneath the nucleic acid-binding channel that appeared to stabilize the mature RISC. Indeed, mutating the water-pocket residues of Argonaute2 and Argonaute4 compromised RISC assembly. Simulations predict that internal water molecules are exchangeable with the bulk solvent but always occupy specific positions at the domain interfaces. These results suggest that after guide RNA-driven conformational changes, water-mediated hydrogen-bonding networks tie together the converged domains to complete the functional RISC structure.

High-Throughput Analysis Reveals Rules for Target RNA Binding and Cleavage by AGO2.

Argonaute proteins loaded with microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC), which represses target RNA expression. Predicting the biological targets, specificity, and efficiency of both miRNAs and siRNAs has been hamstrung by an incomplete understanding of the sequence determinants of RISC binding and cleavage. We applied high-throughput methods to measure the association kinetics, equilibrium binding energies, and single-turnover cleavage rates of mouse AGO2 RISC. We find that RISC readily tolerates insertions of up to 7 nt in its target opposite the central region of the guide. Our data uncover specific guide:target mismatches that enhance the rate of target cleavage, suggesting novel siRNA design strategies. Using these data, we derive quantitative models for RISC binding and target cleavage and show that our in vitro measurements and models predict knockdown in an engineered cellular system.

RNA-induced initiation of transcriptional silencing (RITS) complex structure and function.

Heterochromatic regions of the genome are epigenetically regulated to maintain a heritable '"silent state"'. In fission yeast and other organisms, epigenetic silencing is guided by nascent transcripts, which are targeted by the RNA interference pathway. The key effector complex of the RNA interference pathway consists of small interfering RNA molecules (siRNAs) associated with Argonaute, assembled into the RNA-induced transcriptional silencing (RITS) complex. This review focuses on our current understanding of how RITS promotes heterochromatin formation, and in particular on the role of Argonaute-containing complexes in many other functions such as quelling, release of RNA polymerases, cellular quiescence and genome defense.

The Role of Tertiary Structure in MicroRNA Target Recognition.

Translational repression and degradation of transcripts by microRNAs (miRNAs) is mediated by a ribonucleoprotein complex called the miRNA-induced silencing complex (miRISC, or RISC). Advances in experimental determination of RISC structures have enabled detailed analysis and modeling of known miRNA targets, yet a full appreciation of the structural factors influencing target recognition remains a challenge, primarily because target recognition involves a combination of RNA-RNA and RNA-protein interactions that can vary greatly among different miRNA-target pairs. In this chapter, we review progress toward understanding the role of tertiary structure in miRNA target recognition using computational approaches to assemble RISC complexes at known targets and physics-based methods for computing target interactions. Using this framework to examine RISC structures and dynamics, we describe how the conformational flexibility of Argonautes plays an important role in accommodating the diversity of miRNA-target duplexes formed at canonical and noncanonical target sites. We then discuss applications of tertiary structure-based approaches to emerging topics, including the structural effects of SNPs in miRNA targets and cooperative interactions involving Argonaute-Argonaute complexes. We conclude by assessing the prospects for genome-scale modeling of RISC structures and modeling of higher-order Argonaute complexes associated with miRNA biogenesis, mRNA regulation, and other functions.

CD95/Fas ligand mRNA is toxic to cells.

CD95/Fas ligand binds to the death receptor CD95 to induce apoptosis in sensitive cells. We previously reported that CD95L mRNA is enriched in sequences that, when converted to si/shRNAs, kill all cancer cells by targeting critical survival genes (Putzbach et al., 2017). We now report expression of full-length CD95L mRNA itself is highly toxic to cells and induces a similar form of cell death. We demonstrate that small (s)RNAs derived from CD95L are loaded into the RNA induced silencing complex (RISC) which is required for the toxicity and processing of CD95L mRNA into sRNAs is independent of both Dicer and Drosha. We provide evidence that in addition to the CD95L transgene a number of endogenous protein coding genes involved in regulating protein translation, particularly under low miRNA conditions, can be processed to sRNAs and loaded into the RISC suggesting a new level of cell fate regulation involving RNAi.

Structural insights into Drosophila-C3PO complex assembly and 'Dynamic Side Port' model in substrate entry and release.

In Drosophila and human, component 3 promoter of RISC (C3PO), a heteromeric complex, enhances RISC assembly and promotes RISC activity. Here, we report crystal structure of full-length Drosophila C3PO (E126Q), an inactive C3PO mutant displaying much weaker RNA binding ability, at 2.1 Å resolution. In addition, we also report the cryo-EM structures of full-length Drosophila C3PO (E126Q), C3PO (WT) and SUMO-C3PO (WT, sumo-TRAX + Translin) particles trapped at different conformations at 12, 19.7 and 12.8 Å resolutions, respectively. Crystal structure of C3PO (E126Q) displays a half-barrel architecture consisting of two Trax/Translin heterodimers, whereas cryo-EM structures of C3PO (E126Q), C3PO (WT) and SUMO-C3PO (WT) adopt a closed football-like shape with a hollow interior cavity. Remarkably, both cryo-EM structures of Drosophila C3PO (E126Q) and Drosophila SUMO-C3PO (WT) particles contain a wide side port (∼25 Å × âˆ¼30 Å versus ∼15 Å × âˆ¼20 Å) for RNA substrate entry and release, formed by a pair of anti-parallel packed long α1 helices of TRAX subunits. Notably, cryo-EM structure of SUMO-C3PO showed that four copies of extra densities belonging to N-terminal SUMO tag are located at the outside shell of SUMO-C3PO particle, which demonstrated that the stoichiometry of TRAX/Translin for the in vitro expressed and assembled full-length Drosophila-SUMO-C3PO particle is 4:4, suggesting Drosophila C3PO is composed by TRAX/translin at a ratio of 4:4. Remarkably, the comparison of the cryo-EM structures suggests that the C3PO side ports regulated by α1 helices of TRAX molecules are highly dynamic. Hence, we propose that C3PO particles could adopt a 'Dynamic Side Port' model to capture/digest nucleic acid duplex substrate and release the digested fragments through the dynamic side ports.

Structurally modulated codelivery of siRNA and Argonaute 2 for enhanced RNA interference.

Small interfering RNA (siRNA) represents a promising class of inhibitors in both fundamental research and the clinic. Numerous delivery vehicles have been developed to facilitate siRNA delivery. Nevertheless, achieving highly potent RNA interference (RNAi) toward clinical translation requires efficient formation of RNA-induced gene-silencing complex (RISC) in the cytoplasm. Here we coencapsulate siRNA and the central RNAi effector protein Argonaute 2 (Ago2) via different delivery carriers as a platform to augment RNAi. The physical clustering between siRNA and Ago2 is found to be indispensable for enhanced RNAi. Moreover, by utilizing polyamines bearing the same backbone but distinct cationic side-group arrangements of ethylene diamine repeats as the delivery vehicles, we find that the molecular structure of these polyamines modulates the degree of siRNA/Ago2-mediated improvement of RNAi. We apply this strategy to silence the oncogene STAT3 and significantly prolong survival in mice challenged with melanoma. Our findings suggest a paradigm for RNAi via the synergistic coassembly of RNA with helper proteins.

In vitro reconstitution of chaperone-mediated human RISC assembly.

To silence target mRNAs, small RNAs and Argonaute (Ago) proteins need to be assembled into RNA-induced silencing complexes (RISCs). Although the assembly of Drosophila melanogaster RISC was recently reconstituted by Ago2, the Dicer-2/R2D2 heterodimer, and five chaperone proteins, the absence of a reconstitution system for mammalian RISC assembly has posed analytical challenges. Here we describe reconstitution of human RISC assembly using Ago2 and five recombinant chaperone proteins: Hsp90ß, Hsc70, Hop, Dnaja2, and p23. Our data show that ATP hydrolysis by both Hsp90ß and Hsc70 is required for RISC assembly of small RNA duplexes but not for that of single-stranded RNAs. The reconstitution system lays the groundwork for further studies of small RNA-mediated gene silencing in mammals.

Regulation of the activity of the promoter of RNA-induced silencing, C3PO.

RNA-induced silencing is a process which allows cells to regulate the synthesis of specific proteins. RNA silencing is promoted by the protein C3PO (component 3 of RISC). We have previously found that phospholipase Cß, which increases intracellular calcium levels in response to specific G protein signals, inhibits C3PO activity towards certain genes. Understanding the parameters that control C3PO activity and which genes are impacted by G protein activation would help predict which genes are more vulnerable to downregulation. Here, using a library of 1018 oligonucleotides, we show that C3PO binds oligonucleotides with structural specificity but little sequence specificity. Alternately, C3PO hydrolyzes oligonucleotides with a rate that is sensitive to substrate stability. Importantly, we find that oligonucleotides with higher Tm values are inhibited by bound PLCß. This finding is supported by microarray analysis in cells over-expressing PLCß1. Taken together, this study allows predictions of the genes whose post-transcriptional regulation is responsive to the G protein/phospholipase Cß/calcium signaling pathway.

5΄-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo.

5΄-Vinylphosphonate modification of siRNAs protects them from phosphatases, and improves silencing activity. Here, we show that 5΄-vinylphosphonate confers novel properties to siRNAs. Specifically, 5΄-vinylphosphonate (i) increases siRNA accumulation in tissues, (ii) extends duration of silencing in multiple organs and (iii) protects siRNAs from 5΄-to-3΄ exonucleases. Delivery of conjugated siRNAs requires extensive chemical modifications to achieve stability in vivo. Because chemically modified siRNAs are poor substrates for phosphorylation by kinases, and 5΄-phosphate is required for loading into RNA-induced silencing complex, the synthetic addition of a 5΄-phosphate on a fully modified siRNA guide strand is expected to be beneficial. Here, we show that synthetic phosphorylation of fully modified cholesterol-conjugated siRNAs increases their potency and efficacy in vitro, but when delivered systemically to mice, the 5΄-phosphate is removed within 2 hours. The 5΄-phosphate mimic 5΄-(E)-vinylphosphonate stabilizes the 5΄ end of the guide strand by protecting it from phosphatases and 5΄-to-3΄ exonucleases. The improved stability increases guide strand accumulation and retention in tissues, which significantly enhances the efficacy of cholesterol-conjugated siRNAs and the duration of silencing in vivo. Moreover, we show that 5΄-(E)-vinylphosphonate stabilizes 5΄ phosphate, thereby enabling systemic delivery to and silencing in kidney and heart.

A non-canonical site reveals the cooperative mechanisms of microRNA-mediated silencing.

Although strong evidence supports the importance of their cooperative interactions, microRNA (miRNA)-binding sites are still largely investigated as functionally independent regulatory units. Here, a survey of alternative 3΄UTR isoforms implicates a non-canonical seedless site in cooperative miRNA-mediated silencing. While required for target mRNA deadenylation and silencing, this site is not sufficient on its own to physically recruit miRISC. Instead, it relies on facilitating interactions with a nearby canonical seed-pairing site to recruit the Argonaute complexes. We further show that cooperation between miRNA target sites is necessary for silencing in vivo in the C. elegans embryo, and for the recruitment of the Ccr4-Not effector complex. Using a structural model of cooperating miRISCs, we identified allosteric determinants of cooperative miRNA-mediated silencing that are required for both embryonic and larval miRNA functions. Our results delineate multiple cooperative mechanisms in miRNA-mediated silencing and further support the consideration of target site cooperation as a fundamental characteristic of miRNA function.

Kinetic Analysis of Target RNA Binding and Slicing by Human Argonaute 2 Protein.

Analyzing the mechanisms of Argonaute-mediated gene silencing is essential to the understanding of RNA interference (RNAi). RNAi is a process to regulate gene expression on a posttranscriptional level. Directed by single-stranded small RNA guides, Argonaute 2 binds complementary target RNAs, and if the guide displays full complementarity to the targeted sequence, Argonaute 2 slices the bound target RNA. This on the one hand is an important mechanism to regulate gene expression in the cell and on the other hand represents a powerful tool to interfere with harmful gene expression levels. Here, we present techniques to kinetically characterize recombinant Argonaute 2-mediated guide and target binding as well as target RNA slicing. We focus on fluorescence-based steady-state and in particular pre-steady-state techniques to unravel mechanistic details. Furthermore, we describe a cleavage assay to analyze Argonaute 2-mediated slicing using radioactively labeled target strands.