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Posttranscriptional gene silencing is mediated by RNA‐induced silencing complexes (RISCs) that contain AGO proteins and single‐stranded small RNAs. The assembly of plant AGO1‐containing RISCs depends on the molecular chaperone HSP90. Here, we demonstrate that cyclophilin 40 (CYP40), protein phosphatase 5 (PP5), and several other proteins with the tetratricopeptide repeat (TPR) domain associates with AGO1 in an HSP90‐dependent manner in extracts of evacuolated tobacco protoplasts (BYL). Intriguingly, CYP40, but not the other TPR proteins, could form a complex with small RNA duplex‐bound AGO1. Moreover, CYP40 that was synthesized by in‐vitro translation using BYL uniquely facilitated binding of small RNA duplexes to AGO1, and as a result, increased the amount of mature RISCs that could cleave target RNAs. CYP40 was not contained in mature RISCs, indicating that the association is transient. Addition of PP5 or cyclophilin‐binding drug cyclosporine A prevented the association of endogenous CYP40 with HSP90–AGO1 complex and inhibited RISC assembly. These results suggest that a complex of AGO1, HSP90, CYP40, and a small RNA duplex is a key intermediate of RISC assembly in plants. AGO1 plays an important role in the miRNA pathway in plants; however, relatively little is known about the assembly of the AGO1–miRISC complex in this organism. This study shows that cyclophilin 40 promotes the association of HSP90–AGO1 with duplexed small RNAs, while PP5 opposes this effect.

Mitochondria are important organelles in cellular metabolism. Several crucial metabolic pathways such as the energy producing electron transport chain or the tricarboxylic acid cycle are hosted inside the mitochondria. The proper function of mitochondria depends on the import of proteins, which are encoded in the nucleus and synthesized in the cytosol. Micro-ribonucleic acids (miRNAs) are short non-coding ribonucleic acid (RNA) molecules with the ability to prevent messenger RNA (mRNA)-translation or to induce the degradation of mRNA-transcripts. Although miRNAs are mainly located in the cytosol or the nucleus, a subset of ~150 different miRNAs, called mitomiRs, has also been found localized to mitochondrial fractions of cells and tissues together with the subunits of the RNA-induced silencing complex (RISC); the protein complex through which miRNAs normally act to prevent translation of their mRNA-targets. The focus of this review is on miRNAs and mitomiRs with influence on mitochondrial metabolism and their possible pathophysiological impact.

MicroRNAs in Parkinson's Mutations in leucine-rich repeat kinase 2 (LRRK2) have been linked to both familial and sporadic Parkinson's disease, but the biochemical function of LRRK2 has remained elusive. Now that function has been discovered. Both Drosophila and human LRRK2 are shown to antagonize microRNA-mediated translational inhibition of E2F1 and DP transcription factors. LRRK2 interacts with the RNA-induced silencing complex component Argonaute to antagonize its suppressive effect on protein translation. In vivo genetic studies demonstrate a key role for E2F1/DP upregulation in mediating mutant LRRK2 pathogenesis. These findings point to a link between impaired microRNA-mediated silencing and deregulated expression of specific microRNA targets to Parkinson's disease pathogenesis, and suggest possible microRNA-based therapeutic approaches. LRRK2 activity is dysregulated in Parkinson's disease, but how it contributes to the pathogenesis is unknown. These authors show that Drosophila LRRK2 interacts with the Argonaute component (dAgo1) of the RNA-induced silencing complex. This is associated with reduced levels of dAgo1, interaction between phospho-4E-BP1 and hAgo2, and impairment of microRNA-mediated repression. This leads to overexpression of the cell cycle genes e2f1 and dp , and consequent degeneration of dopaminergic neurons. Gain-of-function mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial as well as sporadic Parkinson’s disease characterized by age-dependent degeneration of dopaminergic neurons 1 , 2 . The molecular mechanism of LRRK2 action is not known. Here we show that LRRK2 interacts with the microRNA (miRNA) pathway to regulate protein synthesis. Drosophila e2f1 and dp messenger RNAs are translationally repressed by let-7 and miR-184*, respectively. Pathogenic LRRK2 antagonizes these miRNAs, leading to the overproduction of E2F1/DP, previously implicated in cell cycle and survival control 3 and shown here to be critical for LRRK2 pathogenesis. Genetic deletion of let-7, antagomir-mediated blockage of let-7 and miR-184* action, transgenic expression of dp target protector, or replacement of endogenous dp with a dp transgene non-responsive to let-7 each had toxic effects similar to those of pathogenic LRRK2. Conversely, increasing the level of let-7 or miR-184* attenuated pathogenic LRRK2 effects. LRRK2 associated with Drosophila Argonaute-1 (dAgo1) or human Argonaute-2 (hAgo2) of the RNA-induced silencing complex (RISC). In aged fly brain, dAgo1 protein level was negatively regulated by LRRK2. Further, pathogenic LRRK2 promoted the association of phospho-4E-BP1 with hAgo2. Our results implicate deregulated synthesis of E2F1/DP caused by the miRNA pathway impairment as a key event in LRRK2 pathogenesis and suggest novel miRNA-based therapeutic strategies.

In a phase 1 trial, healthy volunteers were assigned to an RNAi therapeutic inhibitor of PCSK9 or placebo. Single doses of 300 mg or more reduced LDL cholesterol by up to 50%; multiple-dose regimens reduced LDL cholesterol by up to 59%. No serious adverse events were reported. An elevated level of low-density lipoprotein (LDL) cholesterol is a major risk factor for cardiovascular disease. 1 Despite the use of statin therapy, alone or in combination with other lipid-lowering medications, many at-risk patients continue to have elevated levels of LDL cholesterol. 2 – 4 Hence, there is a need for additional treatment options for lowering of the LDL cholesterol level to reduce cardiovascular risk. Proprotein convertase subtilisin–kexin type 9 (PCSK9) is a recently identified but well-validated target for LDL cholesterol–lowering therapy. 5 This serine protease, which is expressed and secreted into the bloodstream predominantly by the liver, binds LDL receptors both intracellularly and . . .

The slicer activity of the RNA-induced silencing complex resides within its Argonaute (Ago) component, in which the PIWI domain provides the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. Here we report on structures of ternary complexes of Thermus thermophilus Ago catalytic mutants with 5′-phosphorylated 21-nucleotide guide DNA and complementary target RNAs of 12, 15 and 19 nucleotides in length, which define the molecular basis for Mg 2+ -facilitated site-specific cleavage of the target. We observe pivot-like domain movements within the Ago scaffold on proceeding from nucleation to propagation steps of guide–target duplex formation, with duplex zippering beyond one turn of the helix requiring the release of the 3′-end of the guide from the PAZ pocket. Cleavage assays on targets of various lengths supported this model, and sugar-phosphate-backbone-modified target strands showed the importance of structural and catalytic divalent metal ions observed in the crystal structures. How Argonaute slices mRNA One of the means by which RNA silencing downregulates gene expression is through small RNA-directed cleavage of a complementary mRNA. Argonaute family proteins are essential regulators of gene expression in plants and animals. They act by binding to single-stranded guide RNA molecules to form RISC complexes that catalyse splitting of the mRNA just where the sequence is complementary to the Argonaute-bound RNA. The crystal structure of several Thermus thermophilus Argonaute proteins mutated to inhibit cleavage activity have now been determined, in complex with a guide RNA and target RNAs of different lengths. Comparison of the various structures reveals conformational changes as the guide and target RNAs pair and suggests a mechanism for cleavage involving three aspartates and two Mg 2+ ions. The Argonaute (Ago) family of proteins provides the slicer activity of the RNA-induced silencing complex, with the Ago component of the complex providing the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. Here, the crystal structures of ternary complexes of Thermus thermophilus Ago catalytic mutants are reported and analysed.

Here we report on a 3.0 Å crystal structure of a ternary complex of wild-type Thermus thermophilus argonaute bound to a 5′-phosphorylated 21-nucleotide guide DNA and a 20-nucleotide target RNA containing cleavage-preventing mismatches at the 10–11 step. The seed segment (positions 2 to 8) adopts an A-helical-like Watson–Crick paired duplex, with both ends of the guide strand anchored in the complex. An arginine, inserted between guide-strand bases 10 and 11 in the binary complex, locking it in an inactive conformation, is released on ternary complex formation. The nucleic-acid-binding channel between the PAZ- and PIWI-containing lobes of argonaute widens on formation of a more open ternary complex. The relationship of structure to function was established by determining cleavage activity of ternary complexes containing position-dependent base mismatch, bulge and 2′- O -methyl modifications. Consistent with the geometry of the ternary complex, bulges residing in the seed segments of the target, but not the guide strand, were better accommodated and their complexes were catalytically active. RNAi: the Argonaute ternary complex Argonaute or 'Ago' proteins are the 'slicer' components of RISC, the RNA-induced silencing protein complex at the heart of the gene silencing mechanism known as RNA interference (RNAi). The structure of a thermophilic Ago protein bound to a duplex nucleic acid that mimics the interaction of the single-stranded small RNA and the target mRNA has now been determined. The structure reveals that the catalytic cycle is driven by conformational changes in both Argonaute and its bound template. The structure of a thermophilic Ago protein bound to a duplex nucleic acid that mimics the interaction of the single-strand of the small RNA and the target mRNA has been solved. This structure reveals the conformational changes that are necessary to accommodate the target, and the changes that occur in the vicinity of the site of cleavage.

Small RNAs function within the context of RNA-induced silencing complexes (RISCs) containing Argonaute (AGO) subfamily proteins. Experiments now show that human RISC assembly is uncoupled from dicing and is facilitated by ATP to load the small RNA duplexes but is not necessary to unwind them. The four human AGO proteins show no obvious structural preferences for small RNA duplexes in contrast to the situation in flies and worms where small RNAs are actively sorted into distinct AGO proteins according to their structural features. The assembly of RNA-induced silencing complex (RISC) is a key process in small RNA–mediated gene silencing. In humans, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are incorporated into RISCs containing the Argonaute (AGO) subfamily proteins Ago1–4. Previous studies have proposed that, unlike Drosophila melanogaster RISC assembly pathways, human RISC assembly is coupled with dicing and is independent of ATP. Here we show by careful reexamination that, in humans, RISC assembly and dicing are uncoupled, and ATP greatly facilitates RISC loading of small-RNA duplexes. Moreover, all four human AGO proteins show remarkably similar structural preferences for small-RNA duplexes: central mismatches promote RISC loading, and seed or 3′-mid (guide position 12–15) mismatches facilitate unwinding. All these features of human AGO proteins are highly reminiscent of fly Ago1 but not fly Ago2.

MicroRNAs (miRNAs) are ∼22 nt long, non‐coding RNAs that guide post‐transcriptional gene silencing of their target genes and regulate diverse biological processes including cancer. miRNAs do not act alone, but require assembly into RNA‐induced silencing complex (RISC). In this review, we summarize how miRNAs are produced, assembled into RISC, and regulate target mRNAs, and discuss how the miRNA pathway is involved in cancer. (Cancer Sci 2010; 101: 2309–2315)

Small RNAs such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) silence the expression of their complementary target messenger RNAs 1 , 2 via the formation of effector RNA-induced silencing complexes (RISCs), which contain Argonaute (Ago) family proteins at their core. Although loading of siRNA duplexes into Drosophila Ago2 requires the Dicer-2–R2D2 heterodimer 3 , 4 , 5 and the Hsc70/Hsp90 (Hsp90 also known as Hsp83) chaperone machinery 6 , 7 , 8 , the details of RISC assembly remain unclear. Here we reconstitute RISC assembly using only Ago2, Dicer-2, R2D2, Hsc70, Hsp90, Hop, Droj2 (an Hsp40 homologue) and p23. By following the assembly of single RISC molecules, we find that, in the absence of the chaperone machinery, an siRNA bound to Dicer-2–R2D2 associates with Ago2 only transiently. The chaperone machinery extends the dwell time of the Dicer-2–R2D2–siRNA complex on Ago2, in a manner dependent on recognition of the 5′-phosphate on the siRNA guide strand. We propose that the chaperone machinery supports a productive state of Ago2, allowing it to load siRNA duplexes from Dicer-2–R2D2 and thereby assemble RISC. The assembly of single Drosophila RNA-induced silencing complexes (RISCs) is reconstituted using seven purified proteins, revealing that chaperones help stabilize the interaction of the protein heterodimer Dicer-2–R2D2 bound to the short interfering RNA with Ago2. A functioning RISC complex reconstituted The central effector involved in RNA silencing is RISC, the RNA-induced silencing complex, composed of an Argonaute (Ago) protein bound to a small interfering RNA (siRNA). Yukihide Tomari and colleagues have now reconstituted the assembly of single Drosophila RISC molecules using eight purified proteins. They find that the chaperones help to stabilize the interaction of the siRNA, which is initially bound to a complex of Dicer-2 and R2D2, with Ago2. This stabilization allows Ago2 to recognize the 5′-phosphate of the siRNA guide strand, after which the guide strand is transferred onto Ago2.

The RNA interference (RNAi) machinery is a key cellular mechanism catalyzing biogenesis and function of miRNAs to post-transcriptionally regulate mRNA expression. The RNAi machinery includes a set of protein complexes with subcellular localization traditionally presented in a uniform fashion: the microprocessor processes miRNAs in the nucleus, whereas the DICER and the RNA-induced silencing complex (RISC) further process and enable activity of miRNAs in the cytoplasm. However, several studies have identified subcellular patterns of RNAi components that deviate from this model. We have particularly shown that RNAi complexes associate with the adherens junctions of well-differentiated epithelial cells, through the E-cadherin partner PLEKHA7. To assess the extent of these subcellular topological patterns, we examined subcellular localization of the microprocessor and RISC in a series of human cell lines and normal human tissues. Our results show that junctional localization of RNAi components is a broad characteristic of differentiated epithelia, but it is absent in transformed or mesenchymal cells and tissues. We also find extensive localization of the microprocessor in the cytoplasm, as well as of RISC in the nucleus. These findings expose a RNAi machinery with multifaceted subcellular topology that may inform its physiological role and calls for updating of the current models.