The solution structures of the Argonaute2 PAZ domain bound to RNA and DNA oligonucleotides is described. The structures reveal a unique mode of single-stranded nucleic acid binding in which the two 3'-terminal nucleotides are buried in a hydrophobic cleft. It is proposed that the PAZ domain contributes to the specific recognition of siRNAs by providing a binding pocket for their characteristic two-nucleotide 3' overhangs (Lingel, 2004).
Argonaute proteins associate with small RNAs that guide mRNA degradation, translational repression, or a combination of both. The human Argonaute family has eight members, four of which (Ago1 through Ago4) are closely related and coexpressed in many cell types. To understand the biological function of the different Ago proteins, attempts were made to determine if Ago1 through Ago4 are associated with miRNAs as well as RISC activity in human cell lines. The results suggest that miRNAs are incorporated indiscriminately of their sequence into Ago1 through Ago4 containing microRNPs (miRNPs). Purification of the FLAG/HA-epitope-tagged Ago containing complexes from different human cell lines revealed that endonuclease activity is exclusively associated with Ago2. Exogenously introduced siRNAs also associate with Ago2 for guiding target RNA cleavage. The specific role of Ago2 in guiding target RNA cleavage was confirmed independently by siRNA-based depletion of individual Ago members in combination with a sensitive positive-readout reporter assay (Meister, 2004).
Argonaute proteins and small interfering RNAs (siRNAs) are the known signature components of the RNA interference effector complex RNA-induced silencing complex (RISC). However, the identity of 'Slicer', the enzyme that cleaves the messenger RNA (mRNA) as directed by the siRNA, has not been resolved. The crystal structure of the Argonaute protein from Pyrococcus furiosus is reported at 2.25Å resolution. The structure reveals a crescent-shaped base made up of the amino-terminal, middle, and PIWI domains. The Piwi Argonaute Zwille (PAZ) domain is held above the base by a 'stalk'-like region. The PIWI domain (named for the protein piwi) is similar to ribonuclease H, with a conserved active site aspartate-aspartate-glutamate motif, strongly implicating Argonaute as 'Slicer'. The architecture of the molecule and the placement of the PAZ and PIWI domains define a groove for substrate binding and suggest a mechanism for siRNA-guided mRNA cleavage (Song, 2004).
RNA silencing regulates gene expression through mRNA degradation, translation repression and chromatin remodelling. The fundamental engines of RNA silencing are RISC and RITS complexes, whose common components are 21-25 nt RNA and an Argonaute protein containing a PIWI domain of unknown function. The crystal structure of an archaeal Piwi protein (AfPiwi) is organised into two domains, one resembling the sugar-binding portion of the lac repressor and another with similarity to RNase H. Invariant residues and a coordinated metal ion lie in a pocket that surrounds the conserved C-terminus of the protein, defining a key functional region in the PIWI domain. Furthermore, two Asp residues, conserved in the majority of Argonaute sequences, align spatially with the catalytic Asp residues of RNase H-like catalytic sites, suggesting that in eukaryotic Argonaute proteins the RNase H-like domain may possess nuclease activity. The conserved region around the C-terminus of the PIWI domain, which is required for small interfering RNA (siRNA) binding to AfPiwi, may function as the receptor site for the obligatory 5' phosphate of siRNAs, thereby specifying the cleavage position of the target mRNA (Parker, 2004).
Double-stranded RNA induces potent and specific gene silencing through a process referred to as RNA interference (RNAi) or posttranscriptional gene silencing (PTGS). RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (approximately 22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity are unknown. The RNAi effector nuclease has been purified from cultured Drosophila cells. The active fraction contains a ribonucleoprotein complex of approximately 500 kilodaltons. Protein microsequencing reveals that one constituent of this complex, Argonaute2, is a homolog of genes that are essential for gene silencing in Caenorhabditis elegans, Neurospora, and Arabidopsis. This observation begins the process of forging links between genetic analysis of RNAi from diverse organisms and the biochemical model of RNAi that is emerging from Drosophila in vitro systems (Hammond, 2001).
Posttranscriptional silencing phenomena have also been observed in plants (e.g., PTGS) and fungi (e.g., quelling): genetic studies indicate that these are likely to be mechanistically related to RNAi. Moreover, RNAi per se has been demonstrated in a variety of experimental systems, including insects, protozoans, and mammals. A synthesis of in vivo and in vitro experiments has led to a mechanistic model for RNAi/PTGS. Silencing is initiated by exposure of a cell to dsRNA. This 'trigger' may be introduced experimentally or may derive from endogenous sources such as viruses, transgenes, or cellular genes. Double-stranded RNAs are processed into discrete ~21- to 25-nucleotide (nt) RNA fragments known as siRNAs (small interfering RNAs). These small RNAs join a multicomponent nuclease complex, RISC, and guide that enzyme to its substrates through conventional base-pairing interactions. Recognition of mRNAs by RISC leads to their destruction (Hammond, 2001).
To date, mechanistic studies have approached RNAi/PTGS from two standpoints. Genetic studies have identified nearly a dozen genes that affect the dsRNA response. These include genes that encode putative nucleases (mut-7), helicases (qde-3, SDE3, mut-6), RNA-dependent RNA polymerases (e.g., ego-1, qde-1, SDE1/SGS2), and members of the Argonaute family (rde-1, qde-2, AGO1). Biochemical studies, carried out exclusively in extracts from Drosophila embryos and cultured cells, have identified enzymatic activities that are proposed to contribute to the interference process. However, links between biochemical and genetic studies of RNAi have yet to be made (Hammond, 2001 and references therein).
This study reports attempts to identify the proteins and RNAs that carry out RNAi in vitro as a step toward unifying biochemical and genetic data into a single mechanistic model. A ribonuclease III family enzyme, Dicer, is a candidate for processing long dsRNA silencing triggers into ~22-nt siRNAs. A requirement for Dicer in RNAi in vivo has been demonstrated in C. elegans. This study reports the biochemical purification of RISC, the effector nuclease of RNAi, and the identification of one subunit of this enzyme. This protein is a member of the Argonaute family, which has been linked to RNAi through genetic studies in several experimental systems (Hammond, 2001).
RNA interference can be provoked in cultured Drosophila S2 cells by transfection with dsRNA, or indeed by simply adding dsRNA to the culture media. Extracts from such cells contain a nuclease complex, RISC, that specifically degrades mRNAs that are homologous to the dsRNA trigger. The hypothesis that this nuclease constitutes the effector activity of RNAi is strengthened by the observation that RISC cofractionates with ~22-nt RNAs that are derived directly from the silencing trigger. Furthermore, this nuclease contains an essential nucleic acid subunit, which is presumably a siRNA (Hammond, 2001).
A biochemical fractionation protocol has been developed that permits the purification of RISC to near-homogeneity. RISC is bound to ribosomes in cell-free extracts; however, the biological relevance of this association remains to be established. Ribosomes can be concentrated from S2 lysates by high-speed centrifugation, and soluble RISC can be recovered from the ribosome pellet by extraction with high concentrations of salt (Hammond, 2001).
Size fractionation of soluble RISC yielded a single peak of sequence-specific nuclease activity. Thus, a single complex contains all the activities and information needed to identify and degrade cognate mRNAs. The large size of this complex (~500 kD) is consistent with its being composed of several subunits, which, according to previous studies, comprise both RNA and protein. A series of additional chromatographic steps were developed that yielded a fraction with a sequence-specific nuclease activity that was purified ~1:10,000 from the crude extract (Hammond, 2001).
Analysis of fractions from the hydroxyapatite column by SDS-polyacrylamide gel electrophoresis (PAGE) indicates that the complex has not been purified to complete homogeneity; however, several proteins clearly cofractionate with the active RISC fraction. Candidate proteins were excised from the gel and microsequenced using tandem mass spectroscopy. Two of four bands failed to produce protein sequence. However, numerous peptides were obtained from bands of ~87 and ~130 kD that matched a single Drosophila gene. Database and domain searches identified this peptide as a homolog of rde-1, a member of the Argonaute gene family, which is essential for RNAi in C. elegans. This gene has been named Argonaute2 (AGO2) because of the prior assignment of Argonaute1 to another gene in the Drosophila genome. Although the Drosophila genome contains at least four Argonaute family members -- AGO1, AGO2, Piwi, and Sting, only AGO2 has been idenfied as a component of RISC in S2 cells. However, the possibility that other Drosophila Argonaute family members join the RISC complex in specific tissues or at specific times during development cannot be excluded (Hammond, 2001).
To verify the presence of AGO2 in RISC, AGO2-specific antibodies were generated. Western blotting of chromatography column fractions with affinity-purified anti-AGO2 shows precise cofractionation of a ~130-kD AGO2 protein and the active RISC fraction through each purification step. In addition, the association between AGO2 and other components of RISC, the siRNAs, was tested. A version of AGO2 was constructed that was tagged at its NH2-terminus with both a T7 epitope and polyhistidine. This was expressed in cells in which RNAi had been induced against firefly luciferase. Tagged AGO2 protein cofractionates with endogenous AGO2, and with the active RISC fraction, in the 500-kD size range. RISC was affinity-purified from cell extracts on a polyhistidine-binding resin. Analysis of the imidazole elution profile from this column by Western blotting with a T7 antiserum and by Northern blotting with a luciferase probe indicates cofractionation of the tagged AGO2 and 22-nt siRNAs. Considered together, these data strongly support the hypothesis that AGO2 is a component of RISC (Hammond, 2001).
To test whether AGO2 is essential for RNAi in Drosophila S2 cells, RNA interference was used to suppress endogenous AGO2. Treatment of S2 cells with either of two different ~1000-nt dsRNAs homologous to AGO2 reduces the levels of this protein by a factor of >10. The ability of these cells to carry out RNAi was assessed by transfection with a mixture of firefly and Renilla luciferase expression plasmids (as an internal control) in combination with either a control dsRNA (green fluorescent protein, GFP) or a firefly luciferase dsRNA. Suppression of AGO2 expression correlates with a pronounced reduction in the ability of cells to silence an exogenous reporter by RNAi (Hammond, 2001).
The biochemical function of Argonaute family members is completely unknown. However, one domain of this protein, the PAZ domain, is shared with Dicer, which initiates RNAi by processing dsRNA silencing triggers into siRNAs. The possibility that Dicer and AGO2 might physically interact, perhaps through their shared PAZ domains, was therefore considered. Indeed, endogenous AGO2 can be coimmunoprecipitated with an epitope-tagged version of Dicer protein from transfected S2 cells. Dicer and RISC are biochemically separable, and none of the purified RISC fractions is able to process dsRNA into 22-nt fragments. One possibility is that Dicer is indeed a component of RISC but fails to process dsRNA when present in this complex. However, the current model is that the interaction between AGO2 and Dicer facilitates the incorporation of siRNAs into RISC complexes, which ultimately dissociate from Dicer and target cognate mRNAs for destruction (Hammond, 2001).
Previous genetic studies in three organisms have indicated that Argonaute family members are essential for RNAi/PTGS. The first link between Argonaute proteins and RNAi was shown by the isolation of C. elegans rde-1 in a screen for RNAi-deficient mutants. In Neurospora, another member of the Argonaute family, QDE-2, emerged from a selection of mutants that were defective in a transgene cosuppression phenomenon, termed 'quelling'. The founding member of this family (AGO1) was first identified in Arabidopsis in a screen for mutants with aberrant leaf morphology. Subsequently, ago1 was re-isolated in a screen for plants that were defective in transgene cosuppression (Hammond, 2001).
Argonaute proteins are typically members of multigene families. In Drosophila there are four annotated genes: Sting, Piwi, AGO1, and AGO2. Mutations in three family members (ago1, piwi, and sting) have previously been studied. Piwi is required for maintenance of cell proliferation in both the male and female germ line, and sting mutations produce spermatid defects and male sterility. Ago1 was identified in a screen for mutations in the wingless pathway, and null mutations in this gene cause defects in neurological development (Hammond, 2001 and references therein).
Thus, Argonaute family members have been linked both to gene silencing phenomena and to the control of development in diverse species. The critical question is whether these two roles of Argonaute proteins are mechanistically related. It is already clear that RNAi-related silencing pathways can control the activity of endogenous genetic elements (e.g., transposons). The possibility also exists that these same pathways may control the expression of endogenous protein-coding genes that regulate development. An answer to this question is likely to emerge both from further genetic studies of RNAi pathways and from a search for endogenous targets of RISC that may be identified via its internal RNA guides to substrate selection (Hammond, 2001).
In Drosophila, Fmr1 binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. A Fmr1-associated complex has been isolated that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The Fmr1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. Dmp68 is also required for efficient RNAi. Fmr1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that Fmr1 is part of the RNAi-related apparatus. These findings suggest a model in which the RNAi and Frm1-mediated translational control pathways intersect in Drosophila. The findings also raise the possibility that defects in an RNAi-related machinery may cause human disease (Ishizuka, 2002).
The identification of AGO2 as a Fmr1-interacting protein is particularly striking. AGO2 is a member of the Argonaute gene family and is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNAi in Drosophila. Therefore, the finding that Fmr1 forms a complex in vivo with AGO2 suggests that Fmr1 may function in RNAi. To test this, RNAi was used to suppress the endogenous proteins, much as had been done previously to establish a role for AGO2 in RNAi. Suppression of ribosomal proteins L5 and L11 with specific dsRNAs made S2 cells so sick that their roles in RNAi could not be assessed. However, treatment of S2 cells with dsRNAs homologous to AGO2, Fmr1, or Dmp68 markedly reduces the levels of these proteins. The ability of these cells to carry out RNAi was tested by transfection with enhanced green fluorescent protein (EGFP) expression plasmid in combination with an EGFP dsRNA. Suppression of AGO2 expression correlates with a pronounced reduction in the ability of cells to silence the reporter EGFP by RNAi. Interestingly, RNAi targeting Dmp68 results in inhibition of RNAi in S2 cells. These results suggest that the DEAD-box helicase Dmp68 not only interacts with Fmr1 but is also required for efficient RNAi in S2 cells. Dmp68 is a Drosophila ortholog of human p68, which has been demonstrated to unwind short but not long dsRNAs in an ATP-dependent manner. It is concluded that at least two of the Fmr1-interacting proteins, AGO2 and Dmp68, are required for RNAi in cultured Drosophila S2 cells. In contrast, depletion of Fmr1 did not appear to affect the EGFP silencing. Therefore, although Fmr1 interacts strongly with AGO2 and Dmp68 in vivo, Fmr1 does not appear to be essential for efficient RNAi (Ishizuka, 2002).
Recent work in numerous organisms has shown that RNAi shares features with a developmental gene regulatory mechanism that involves miRNAs. These small RNAs (siRNAs and miRNAs) are thought to be incorporated into silencing complexes that mediate mRNA destruction during RNAi and translational control during development, respectively. Therefore, it is suggested that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation. AGO2 and Dmp68 are essential for RNAi in Drosophila. However, Fmr1 appears to be a translation repressor. Because Fmr1 interacts with AGO2 and Dmp68 in vivo, it was of interest to examine whether Fmr1 is also present in an AGO2- and/or Dmp68-associated complex. To do this, TAP-tagged AGO2 (AGO2-TAP) or Dmp68 (Dmp68-TAP) were expressed in S2 cells. Cytoplasmic lysate of the cells expressing AGO2-TAP or Dmp68-TAP was prepared and subjected to TAP purifications. In reciprocal assays, endogenous Fmr1 and AGO2 were found to associate with each other. In addition, endogenous AGO2 was copurified with AGO2-TAP. Endogenous Fmr1 and AGO2 were also found to be present in the Dmp68-associated complex. Because AGO2 can be coimmunoprecipitated with Dicer, which initiates RNAi by processing dsRNA silencing triggers into siRNAs, and also processing miRNA precursors into mature miRNAs, the possibility was considered that Fmr1 might also interact physically with Dicer. Indeed, endogenous Dicer can be copurified not only with AGO2-TAP but also with Fmr1-TAP, and it was also shown that Fmr1 remains associated with AGO2 after RNAi induction. It is well established that siRNAs associate with AGO2 during RNAi in S2 cells. Therefore, these results indicate that Fmr1 may be a part of RISC. Finally, analogous to the human AGO2 ortholog (eIF2C2)-associated complex that contains a DEAD-box type RNA helicase and miRNAs, it was of interest to test whether miRNAs are also found in AGO2- and/or Fmr1-associated complexes. RNA molecules copurified with AGO2-TAP or Fmr1-TAP were recovered, dissolved on a 15% polyacrylamide denaturing gel, and subjected to Northern blot analysis. A known miRNA, miR-2b, in Drosophila S2 cells could be detected both in the AGO2- and Fmr1-associated complexes. Together, these data show that Fmr1 is present in a complex with components of RNAi and miRNAs in cultured Drosophila S2 cells (Ishizuka, 2002).
The interaction between Fmr1 and AGO2 remains constant before and after RNAi induction, suggesting that Fmr1 is part of RISC during RNAi. However, there is no evidence to support the notion that RISC formation is induced by treatment of S2 cells with dsRNA. As one of the functions of the RNAi apparatus is to silence transposons and repetitive sequences residing naturally in the Drosophila genome, these cells are therefore likely to be full of pre-formed RISC complexes, irrespective of dsRNA treatment. Therefore, it is possible that Fmr1 is part of the pre-formed RISC complexes and remains to be part of the active RISC after ATP-dependent siRNA unwinding (Ishizuka, 2002).
The involvement of another Fmr1-interacting protein, Dmp68, in RNAi further suggests the close association of Fmr1 with RNAi. The p68 RNA helicase was first identified by cross-reaction with a monoclonal antibody that was originally raised against SV40 large T antigen two decades ago. The helicase plays important roles in cell proliferation and organ maturation and belongs to a large family of highly evolutionarily conserved proteins, the so-called DEAD-box family of putative ATPases and helicases. Recent studies have revealed that several RNA helicases, including mut6, SDE3, mut14 , drh-1, and spindle-E are required for RNAi and related posttranscriptional gene silencing (PTGS) pathways. Dmp68 is similar to, but clearly not an ortholog of these proteins. Therefore, Dmp68 is a novel component of RNAi. Because ATP-dependent unwinding of the siRNA duplex remodels the RISC to generate an active RISC in the RNAi pathway, Dmp68 may mediate the unwinding process. It is also conceivable that Dmp68 may be involved in downstream events such as target RNA recognition, as an RNA chaperone or an RNPase (Ishizuka, 2002).
Fifteen Neurospora crassa mutants defective in 'quelling' or transgene-induced gene silencing have been isolated. These quelling-defective mutants (qde) belonging to three complementation groups have provided insights into the mechanism of posttranscriptional gene silencing in N. crassa. The recessive nature of the qde mutations indicates that the encoded gene products act in trans. When qde genes are mutated in a transgenic-induced silenced strain containing many copies of the transgene, the expression of the endogenous gene is maintained despite the presence of transgene sense RNA, the molecule proposed to trigger quelling. Moreover, the qde mutants failed to show quelling when tested with another gene, suggesting that they may be universally defective in transgene-induced gene silencing. As such, qde genes may be involved in sensing aberrant sense RNA and/or targeting/degrading the native mRNA. The qde mutations may be used to isolate the genes encoding the first components of the quelling mechanism. Moreover, these quelling mutants may be important in applied and basic research for the creation of strains able to overexpress a transgene (Cogoni, 1997).
An allelic series of the novel argonaute mutant (ago1-1 to ago1-6) of the herbaceous plant Arabidopsis thaliana has been isolated. The ago1 mutation pleotropically affects general plant architecture. The apical shoot meristem generates rosette leaves and a single stem, but axillary meristems rarely develop. Rosette leaves lack a leaf blade but still show adaxial/abaxial differentiation. Instead of cauline leaves, filamentous structures without adaxial/abaxial differentiation develop along the stem and an abnormal inflorescence bearing infertile flowers with filamentous organs is produced. Two independent T-DNA insertions into the AGO1 locus led to the isolation of two corresponding genomic sequences as well as a complete cDNA. The AGO1 locus was mapped close to the marker mi291a on chromosome 1. Antisense expression of the cDNA results in a partial mutant phenotype. Sense expression causes some transgenic lines to develop goblet-like leaves and petals. The cDNA encodes a putative 115 kDa protein with sequence similarity to translation products of a novel gene family present in nematodes as well as humans. No specific function has been assigned to these genes. Similar proteins are not encoded by the genomes of yeast or bacteria, suggesting that AGO1 belongs to a novel class of genes with a function specific to multicellular organisms (Bohmert, 1998).
Postembryonic development in higher plants is marked by repetitive organ formation via a self-perpetuating stem cell system, the shoot meristem. Organs are initiated at the shoot meristem periphery, while a central zone harbors the stem cells. The ZWILLE (ZLL) gene of Arabidopsis is specifically required to establish the central-peripheral organization of the embryo apex and this step is critical for shoot meristem self-perpetuation. zll mutants correctly initiate expression of the shoot meristem-specific gene SHOOT MERISTEMLESS in early embryos, but fail to regulate its spatial expression pattern at later embryo stages and initiate differentiated structures in place of stem cells. The ZLL gene was isolated by map-based cloning. It encodes a novel protein, and related sequences are highly conserved in multicellular plants and animals but are absent from bacteria and yeast. It is proposed that ZLL relays positional information required to maintain stem cells of the developing shoot meristem in an undifferentiated state during the transition from embryonic development to repetitive postembryonic organ formation (Moussian, 1998).
Several lines of evidence indicate that the adaxial leaf domain possesses a unique competence to form shoot apical meristems. Factors required for this competence are expected to cause a defect in shoot apical meristem formation when inactivated and to be expressed or active preferentially in the adaxial leaf domain. PINHEAD, a member of a family of proteins that includes the translation factor eIF2C, is required for reliable formation of primary and axillary shoot apical meristems. In addition to high-level expression in the vasculature, low-level PINHEAD expression defines a novel domain of positional identity in the plant. This domain consists of adaxial leaf primordia and the meristem. These findings suggest that the PINHEAD gene product may be a component of a hypothetical meristem forming competence factor. Defects are also described in floral organ number and shape, as well as aberrant embryo and ovule development associated with pinhead mutants, thus elaborating on the role of PINHEAD in Arabidopsis development. In addition, embryos doubly mutant for PINHEAD and ARGONAUTE1, a related, ubiquitously expressed family member, fail to progress to bilateral symmetry and do not accumulate the SHOOT MERISTEMLESS protein. Therefore PINHEAD and ARGONAUTE1 together act to allow wild-type growth and gene expression patterns during embryogenesis (Lynn, 1999).
Introduction of transgene DNA may lead to specific degradation of RNAs that are homologous to the transgene transcribed sequence through phenomena named post-transcriptional gene silencing (PTGS) in plants, quelling in fungi, and RNA interference (RNAi) in animals. PTGS, quelling, and RNAi require a set of related proteins (SGS2, QDE-1, and EGO-1, respectively). Arabidopsis mutants impaired in PTGS have been isolated that are affected at the Argonaute1 (AGO1) locus. AGO1 is similar to QDE-2, required for quelling, and RDE-1, required for RNAi. Sequencing of ago1 mutants has revealed one amino acid essential for PTGS that is also present in QDE-2 and RDE-1 in a highly conserved motif. Taken together, these results confirm the hypothesis that these processes derive from a common ancestral mechanism that controls expression of invading nucleic acid molecules at the post-transcriptional level. As opposed to rde-1 and qde-2 mutants, which are viable, ago1 mutants display several developmental abnormalities, including sterility. These results raise the possibility that PTGS, or at least some of its elements, could participate in the regulation of gene expression during development in plants (Fagard, 2000).
Small RNA molecules have been found to be specifically associated with posttranscriptional gene silencing (PTGS) in both plants and animals. Small sense and antisense RNAs are also involved in PTGS in Neurospora crassa. The accumulation of these RNA molecules depends on the presence of functional qde-1 and qde-3 genes, coding for an RNA dependent RNA polymerase and a helicase respectively, previously shown to be essential for gene silencing, but does not depend on a functional qde-2, coding for an Argonaute family protein, indicating that this gene is involved in a downstream step of the gene silencing pathway. Supporting this idea, a purified QDE2 protein complex was found to contain small RNA molecules, suggesting that QDE2 could be part of a small RNA-directed ribonuclease complex involved in sequence-specific mRNA degradation (Catalonotto, 2002).
Transgene-induced post-transcriptional gene silencing (PTGS) results from specific degradation of RNAs that are homologous with the transgene transcribed sequence. This phenomenon, also known as cosuppression in plants and quelling in fungi, resembles RNA interference (RNAi) in animals. Indeed, cosuppression/quelling/RNAi all require related PAZ/PIWI proteins (AGO1/QDE-2/RDE-1), indicating that these mechanisms are related. Unlike Neurospora crassa qde-2 and Caenorhabditis elegans rde-1 mutants, which are morphologically normal, the 24 known Arabidopsis ago1 mutants display severe developmental abnormalities and are sterile. Hypomorphic Arabidopsis ago1 mutants, described in this study, have been isolated, including fertile ones. These hypomorphic ago1 mutants are defective for PTGS, like null sgs2, sgs3, and ago1 mutants, suggesting that PTGS is more sensitive than development to perturbations in AGO1. Conversely, a mutation in ZWILLE/PINHEAD, another member of the Arabidopsis AGO1 gene family, affects development but not PTGS. Similarly, mutations in ALG-1 and ALG-2, two members of the C. elegans RDE-1 gene family, affect development but not RNAi, indicating that the control of PTGS/RNAi and development by PAZ/PIWI proteins can be uncoupled. Finally, hypomorphic ago1 mutants are hypersensitive to virus infection, confirming the hypothesis that in plants PTGS is a mechanism of defense against viruses (Morel, 2002).
In a number of organisms, transgenes containing transcribed inverted repeats (IRs) that produce hairpin RNA can trigger RNA-mediated silencing, which is associated with 21-24 nucleotide small interfering RNAs (siRNAs). In plants, IR-driven RNA silencing also causes extensive cytosine methylation of homologous DNA in both the transgene 'trigger' and any other homologous DNA sequences -- i.e., 'targets'. Endogenous genomic sequences, including transposable elements and repeated elements, are also subject to RNA-mediated silencing. The RNA silencing gene ARGONAUTE4 (AGO4) is required for maintenance of DNA methylation at several endogenous loci and for the establishment of methylation at the FWA gene. Mutation of AGO4 substantially reduces the maintenance of DNA methylation triggered by IR transgenes, but AGO4 loss-of-function does not block the initiation of DNA methylation by IRs. AGO4 primarily affects non-CG methylation of the target sequences, while the IR trigger sequences lose methylation in all sequence contexts. Finally, AGO4 and the DRM methyltransferase genes are shown to be required for maintenance of siRNAs at a subset of endogenous sequences, but AGO4 is not required for the accumulation of IR-induced siRNAs or a number of endogenous siRNAs, suggesting that AGO4 may function downstream of siRNA production (Zilberman, 2004).
The Schizosaccharomyces pombe genome encodes only one of each of the three major classes of proteins implicated in RNA silencing: Dicer (Dcr1), RNA-dependent RNA polymerase (RdRP; Rdp1), and Argonaute (Ago1). These three proteins are required for silencing at centromeres and for the initiation of transcriptionally silent heterochromatin at the mating-type locus. The introduction of a double-stranded RNA (dsRNA) hairpin corresponding to a green fluorescent protein (GFP) transgene triggers classical RNA interference (RNAi) in S. pombe. That is, GFP silencing triggered by dsRNA reflects a change in the steady-state concentration of GFP mRNA, but not in the rate of GFP transcription. RNAi in S. pombe requires dcr1, rdp1, and ago1, but does not require chp1, tas3, or swi6, genes required for transcriptional silencing. Thus, the RNAi machinery in S. pombe can direct both transcriptional and posttranscriptional silencing using a single Dicer, RdRP, and Argonaute protein. These findings suggest that these three proteins fulfill a common biochemical function in distinct siRNA-directed silencing pathways (Sigova, 2004).
This study demonstrates that a dsRNA derived from a hairpin transcript can trigger posttranscriptional silencing of a corresponding mRNA in S. pombe. A similar hairpin transcript, corresponding to the ura4 locus has also been shown (Schramke, 2003) to trigger transcriptional silencing. In both studies, silencing triggered by a hairpin transcript require the RNAi machinery -- Dcr1, Rdp1, and Ago1. Transcriptional silencing, unlike posttranscriptional silencing, requires components of the transcriptional silencing apparatus: Chp1, Tas3, or Swi6. Robust silencing by both pathways requires the chromodomain protein Clr4, which appears to play a role in siRNA biogenesis or stability. Why does the GFP hairpin construct presented in this study trigger exclusively posttranscriptional silencing, whereas the previously studied ura4 hairpin triggered transcriptional silencing? One possible explanation is that the GFP hairpin used here includes an efficiently spliced intron between the two arms of the hairpin. It is presumed that splicing of the intron promotes the accumulation of GFP dsRNA in the cytoplasm. In contrast, the ura4 hairpin construct of Schramke (2003) contains an unspliced spacer sequence between the hairpin arms. Thus, the ura4 hairpin may be localized largely to the nucleus. A difference in subcellular localization might explain the different results obtained by the two studies. Alternatively, silencing of ura4 by the ura4-specific hairpin might comprise a mixture of transcriptional and posttranscriptional silencing. In this case, transcriptional silencing might not occur at the adh1 locus, even if the GFP hairpin-derived siRNAs trigger histone modification, perhaps because the gene is strongly expressed or is in a region of the genome otherwise refractory to heterochromatin formation. Nonetheless, the current data, together with those of Schramke (2003), clearly show that at least two distinct silencing responses can be initiated by a common RNAi machinery, without resorting to specialized forms of Dicer, RdRP, or Argonaute proteins. The demonstration that fission yeast contain a functional RNAi pathway now provides a simplified, genetically tractable model in which to study how the nature of the silencing trigger or of the silencing target determines the silencing pathway evoked -- posttranscriptional or transcriptional (Sigova, 2004).
RNA interference (RNAi) acts on long double-stranded RNAs (dsRNAs) in a variety of eukaryotes to generate small interfering RNAs that target homologous messenger RNA, resulting in their destruction. This process is widely used to 'knock-down' the expression of genes of interest to explore phenotypes. In plants, fission yeast, ciliates, flies and mammalian cells, short interfering RNAs (siRNAs) also induce DNA or chromatin modifications at the homologous genomic locus, which can result in transcriptional silencing or sequence elimination. siRNAs may direct DNA or chromatin modification by siRNA-DNA interactions at the homologous locus. Alternatively, they may act by interactions between siRNA and nascent transcript. In fission yeast (Schizosaccharomyces pombe), chromatin modifications are directed by RNAi only if the homologous DNA sequences are transcribed. Furthermore, transcription by exogenous T7 polymerase is not sufficient. Ago1, a component of the RNAi effector RISC/RITS complex, associates with target transcripts and RNA polymerase II. Truncation of the regulatory carboxy-terminal domain (CTD) of RNA pol II disrupts transcriptional silencing, indicating that, like other RNA processing events, RNAi-directed chromatin modification is coupled to transcription (Schramke, 2005).
Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, C. elegans mutants resistant to dsRNA-mediated interference (RNAi) have been selected. Two loci, rde-1 and rde-4 (Drosophila homolog: r2d2), are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. One natural function of RNAi is transposon silencing (Tabara, 1999).
In Caenorhabditis elegans, the introduction of double-stranded RNA triggers sequence-specific genetic interference (RNAi) that is transmitted to offspring. The inheritance properties associated with this phenomenon were examined. Transmission of the interference effect occurs through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-1 and rde-4 (coding for a Dicer homolog) are required for the formation of this interfering agent but were not needed for interference thereafter. In contrast, the rde-2 and mut-7 (coding for a ribonuclease) genes were required downstream for interference. These findings provide evidence for germ line transmission of an extragenic sequence-specific silencing factor and implicate rde-1 and rde-4 in the formation of the inherited agent (Grishok, 2000).
RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. This work analyzes cleavage of injected dsRNA to produce the initial siRNA population in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for organismal viability or fertility. The results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger is comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduce the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs can circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed (Parrish, 2001).
Posttranscriptional gene silencing in Caenorhabditis elegans results from exposure to double-stranded RNA (dsRNA), a phenomenon designated as RNA interference (RNAi), or from co-suppression, in which transgenic DNA leads to silencing of both the transgene and the endogenous gene. Single-stranded RNA oligomers of antisense polarity can also be potent inducers of gene silencing. As is the case for co-suppression, antisense RNAs act independently of the RNAi genes rde-1 and rde-4 but require the mutator/RNAi gene mut-7 and a putative DEAD box RNA helicase, mut-14. These data favor the hypothesis that gene silencing is accomplished by RNA primer extension using the mRNA as template, leading to dsRNA that is subsequently degraded (Tijsterman, 2002).
Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. The C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. These findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing (Tabara, 2002).
The argonaute protein family provides central components for RNA interference (RNAi) and related phenomena in a wide variety of organisms. A cDNA clone termed BmAGO2 has been isolated from a Bombyx cell that is homologous to Drosophila ARGONAUTE2, the gene encoding a repressive factor for the recombination repair of extrachromosomal double-strand breaks (DSBs). RNAi-mediated silencing of the BmAGO2 sequence markedly increases homologous recombination (HR) repair of DSBs in episomal DNA, but has no effect on that in chromosomes. Moreover, RNAi for BmAGO2 enhances the integration of linearized DNA into a silkworm chromosome via HR. These results suggest that BmAgo2 protein plays an indispensable role in the repression of extrachromosomal DSB repair (Tsukioka, 2006).
The observation that knockdown of the silkworm Argonaute2 homolog gene, BmAGO2, augments the rate of extrachromosomal HR appears to be explicable by two alternative assumptions: (1) BmAgo2 may act as an inhibitor of HR. This inference comes from the notion that Argonaute protein possibly inhibits the binding of HR-related proteins to the strands via a PAZ domain, which can directly bind to substrate DNA; (2) BmAgo2 is needed to discriminate extrachromosomal DNA from chromosomal DNA and represses extrachromosomal HR repair indirectly. The latter possibility is favored, because Argonaute family proteins have been reported to participate in heterochromatin formation. Argonaute1 is one of the components of the RNA-induced initiation of the transcriptional gene silencing (RITS) complex, which is necessary for heterochromatin assembly. Recent studies indicate that RNAi is involved in heterochromatin formation at the centromere and therefore in chromosome segregation. If BmAgo2 plays a role in the extrachromosomal DNA-specific assembly of heterochromatin, in which HR repair of DSBs are repressed, the decrease in BmAgo2 expression would not affect intrachromosomal HR repair, and this was in fact the case as described above (Tsukioka, 2006).
It is reasonable to predict that cells have defense systems for exogenous DNA, e.g., viral DNA. If such exogenous DNA has a homologous sequence to that of a host genome region, HR between these may frequently cause a partial loss of genomic information. Therefore, the cells would acquire mechanisms to repress the HR repair of extrachromosomal DSBs by using an Argonaute protein. Indeed, baculoviruses, DNA viruses widely isolated from lepidopteran insects, often carry DNA transposable elements, such as piggybac and Tc1-like elements, which apparently originate from the cellular genomes and are inserted into infecting baculovirus genomes. These viruses are to be excluded from the HR-related integration pathway leading to the modification of host genomes (Tsukioka, 2006).
Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. The multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, the evidence supports a model in which Argonaute contributes 'Slicer' activity to RISC, providing the catalytic engine for RNAi (Liu, 2004).
The presence of double-stranded RNA (dsRNA) in most eukaryotic cells provokes a sequence-specific silencing response known as RNA interference (RNAi). The dsRNA trigger of this process can be derived from exogenous sources or transcribed from endogenous noncoding RNA genes that produce microRNAs (miRNAs). RNAi begins with the conversion of dsRNA silencing triggers into small RNAs of ~21 to 26 nucleotides (nts) in length. This is accomplished by the processing of triggers by specialized ribonuclease III (RNase III)-family nucleases, Dicer and Drosha. Resulting small RNAs join an effector complex, known as RISC (RNA-induced silencing complex). Silencing by RISC can occur through several mechanisms. In flies, plants, and fungi, dsRNAs can trigger chromatin remodeling and transcriptional gene silencing. RISC can also interfere with protein synthesis, and this is the predominant mechanism used by miRNAs in mammals. However, the best studied mode of RISC action is mRNA cleavage. When programmed with a small RNA that is fully complementary to the substrate RNA, RISC cleaves that RNA at a discrete position, an activity that has been attributed to an unknown RISC component, 'Slicer'. Whether or not RISC cleaves a substrate can be determined by the degree of complementarity between the siRNA and mRNA, since mismatched duplexes are often not processed. However, even for mammalian miRNAs, which normally repress at the level of protein synthesis, cleavage activity can be detected with a substrate that perfectly matches the miRNA sequence. This result prompted the hypothesis that all RISCs are equal, with the outcome of the RISC-substrate interaction being determined largely by the character of the interaction between the small RNA and its substrate (Liu, 2004 and references therein).
RISC contains two signature components. The first is the small RNA, which cofractionates with RISC activity in Drosophila S2 cell extracts, and whose presence correlates with dsRNA-programmed mRNA cleavage in Drosophila embryo lysates. The second is an Argonaute (Ago) protein, which was identified as a component of purified RISC in Drosophila. Additional studies have suggested that Argonautes are also key components of RISC in mammals, fungi, worms, protozoans, and plants (Liu, 2004 and references therein).
Argonautes are often present as multi-protein families and are identified by two characteristic domains, PAZ and PIWI. These proteins mainly segregate into two subfamilies, comprising those that are more similar to either Arabidopsis Argonaute1 or Drosophila Piwi. The Argonaute family was first linked to RNAi through genetic studies in Caenorhabditis elegans, which identified Rde-1 as a gene essential for silencing . Subsequent placement of a Drosophila Argonaute protein in RISC has prompted an exploration of the roles of this protein family. Toward this end, both biochemical and genetic studies of the Ago1 subfamily proteins in mammals have been undertaken (Liu, 2004).
Mammals contain four Argonaute1 subfamily members -- Ago1 to Ago4. Different Argonaute family members in Drosophila preferentially associate with different small RNAs, with Ago1 preferring miRNAs and Ago2 siRNAs. Recent studies of Drosophila melanogaster (Dm) Ago1 and DmAgo2 mutants have strengthened these conclusions. To assess whether mammalian Ago proteins specialized in their interactions with small RNAs, Ago-associated miRNA populations were examined by microarray analysis. Ago1-, Ago2- and Ago3-associated RNAs were hybridized to microarrays that report the expression status of 152 human microRNAs. Patterns of associated RNAs were identical within experimental error in each case. Additionally, each of the tagged Ago proteins associate similarly with a cotransfected siRNA (Liu, 2004).
Previous studies have used tagged siRNAs to affinity purify Argonaute-containing RISC. These preparations, containing mixtures of at least two mammalian Argonautes, were capable of cleaving synthetic mRNAs that were complementary to the tagged siRNA. The ability of purified complexes containing individual Argonaute proteins to catalyze similar cleavages was examined. Unexpectedly, irrespective of the siRNA sequence, only Ago2-containing RISC was able to catalyze cleavage. All three Ago proteins were similarly expressed and bound similar amounts of transfected siRNA (Liu, 2004).
These results demonstrated that mammalian Argonaute complexes are biochemically distinct, with only a single family member being competent for mRNA cleavage. To examine the possibility that Ago proteins might also be biologically specialized, the mouse Ago2 gene was disrupted by targeted insertional mutagenesis. Intercrosses of Ago2 heterozygotes produced only wild-type and heterozygous offspring, strongly suggesting that disruption of Ago2 produced an embryonic-lethal phenotype (Liu, 2004).
Ago2-deficient mice display several developmental abnormalities, beginning approximately halfway through gestation. Both gene-trap and in situ hybridization data of day 9.5 embryos show broad expression of Ago2 in the embryo, with some hot spots of expression in the forebrain, heart, limb buds, and branchial arches. The most prominent phenotype is a defect in neural tube closure, often accompanied by apparent mispatterning of anterior structures, including the forebrain. Roughly half of the embryos display complete failure of neural tube closure in the head region, while all embryos display a wavy neural tube in more caudal regions. Mutant embryos also suffer from apparent cardiac failure. The hearts are enlarged and often accompanied by pronounced swelling of the pericardial cavity. By day 10.5, mutant embryos are severely developmentally delayed compared with wild-type and heterozygous littermates. This large difference in size, like the apparent cardiac failure, may be accounted for by a general nutritional deficiency caused by yolk sac and placental defects; histological analysis reveals abnormalities in these tissues (Liu, 2004).
Not all Argonaute proteins are required for successful mammalian development. Thus, it is unclear why Ago2 should be required for development, while other Ago proteins are dispensable. Ago subfamily members are expressed in overlapping patterns in humans. In situ hybridization demonstrates overlapping expression patterns for Ago2 and Ago3 in mouse embryos. Considered together with the essentially identical patterns of miRNA binding, these results suggest the possibility that the ability of Ago2 to assemble into catalytically active complexes might be critical for mouse development. Although most miRNAs regulate gene expression at the level of protein synthesis, recently miR196 has been demonstrated to cleave the mRNA encoding HoxB8, a developmental regulator. Evolutionary conservation of an essential cleavage-competent RISC in organisms in which miRNAs predominantly act by translational regulation raises the possibility that target cleavage by mammalian miRNAs might be more important and widespread than previously appreciated (Liu, 2004).
Numerous studies have indicated that experimentally triggered RNAi in mammalian cells proceeds through siRNA-directed mRNA cleavage because in many cases, but not all, reiterated binding sites are necessary for repression at the level of protein synthesis. If Ago2 were uniquely capable of assembling into cleavage-competent complexes in mice, then embryos or cells lacking Ago2 might be resistant to experimental RNAi. To address this question, mouse embryo fibroblasts (MEFs) from E10.5 embryos were prepared from Ago2 heterozygous intercrosses. Reverse transcription polymerase chain reaction (RT-PCR) analysis and genotyping revealed that it was possible to obtain wild-type, mutant, and heterozygous MEF populations. Importantly, MEFs also express other Ago proteins, including Ago1 and Ago3. Ago2-null MEFs were unable to repress gene expression in response to an siRNA. This defect could be rescued by the addition of a third plasmid that encoded human Ago2 but not by a plasmid encoding human Ago1 . In contrast, responses were intact for a reporter of repression at the level of protein synthesis, mediated by an siRNA binding to multiple mismatched sites (Liu, 2004).
Because Ago2 is exceptional in its ability to form cleavage-competent complexes, the determinants of this capacity were mapped. Deletion analysis indicated that an intact Ago2 was required for RISC activity. The sequence of highly conserved but cleavage-incompetent Ago proteins was used as a guide to the construction of Ago2 mutants. A series of point mutations were tested, included H634P, H634A, Q633R, Q633A, H682Y, L140W, F704Y, and T744Y. Whereas all of these mutations retain siRNA-binding activity and most retain cleavage activity, changes at Q633 and H634 have a profound effect on target cleavage. Both the Q633R and H634P mutations, in which residues were changed to corresponding residues in Ago1 and Ago3, abolished catalysis. Changing H634 to A634 also inactivated Ago2, whereas a similar change, Q633A, was permissive for cleavage. Thus, even relatively conservative changes can negate the ability of Ago2 to form cleavage-competent RISC (Liu, 2004).
Several possibilities could explain a lack of cleavage activity for Ago2 mutants. Such mutations could interfere with the proper folding of Ago2. However, this seems unlikely because those same residues presumably permit proper folding in closely related Argonaute proteins, and mutant Ago2 proteins retained the ability to interact with siRNAs. Alternatively, cleavage-incompetent Ago2 mutants could lose the ability to interact with the putative Slicer. Finally, Ago2 itself might be Slicer, with the conservative substitutions altering the active center of the enzyme in a way that prevents cleavage (Liu, 2004).
The last possibility predicted that an active enzyme might be reconstituted with relatively pure Ago2 protein. Ago2 was immunoaffinity purified from 293T cells and attempts were made to reconstitute RISC in vitro. Incubation with the double-stranded siRNA produced no appreciable activity, whereas Ago2 could be successfully programmed with single-stranded siRNAs to cleave a complementary substrate. Formation of the active enzyme was unaffected by first washing the immunoprecipitates with up to 2.5 M NaCl or 1 M urea. A 21-nt single-stranded DNA was unable to direct cleavage. Programming could be accomplished with different siRNAs that direct activity against different substrates. RISC is formed though a concerted assembly process in which the RISC-loading complex (RLC) acts in an adenosine triphosphate (ATP)-dependent manner to place one strand of the small RNA into RISC. In vitro reconstitution occurs in the absence of ATP; this suggests that Ago2 could be programmed with siRNAs without a need for the normal assembly process. However, in vitro reconstitution of RISC still requires the essential characteristics of an siRNA. For example, single-stranded siRNAs that lack a 5' phosphate group cannot reconstitute an active enzyme (Liu, 2004).
Although consistent with the possibility that the catalytic activity of RISC is carried within Ago2, these results do not rule out the possibility that a putative Slicer copurifies with Ago2. To demonstrate more conclusively that Ago2 is Slicer, the crystal structure of an Argonaute protein from an archebacterium, Pyrococcus furiosus, was examined. This structure revealed that the PIWI domain folds into a structure analogous to the catalytic domain of RNase H and avian sarcoma virus (ASV) integrase. The notion that such a domain would lie at the center of RISC cleavage is consistent with previous observations. RNase H and integrases cleave their substrates, leaving 5' phosphate and 3' hydroxyl groups through a metal-catalyzed cleavage reaction. Notably, previous studies have strongly indicated that the scissile phosphate in the targeted mRNA is cleaved via a metal ion in RISC to give the same phosphate polarity. In vitro data are consistent with the reconstituted RISC also requiring a divalent metal (Liu, 2004}.
The active center of RNase H and its relatives consists of a catalytic triad of three carboxylate groups contributed by aspartic or glutamic acid. These amino acid residues coordinate the essential metal and activate water molecules for nucleolytic attack. Reference to the known structure of RNase H reveals two aspartate residues in the archeal Ago protein present at the precise spatial locations predicted for formation of an RNase H-like active site. These align with identical residues in the human Ago2 protein. Therefore, to test whether the PIWI domain of Ago2 provides catalytic activity to RISC, the two conserved aspartates, D597 and D669, were changed to alanine, with the prediction that either mutation would inactivate RISC cleavage. Consistent with the hypothesis, the mutant Ago2 proteins were incapable of assembling into a cleavage-competent RISC in vitro or in vivo, despite retaining the ability to bind siRNAs (Liu, 2004).
Considered together, these data provide strong support for the notion that Argonaute proteins are the catalytic components of RISC: (1) The ability to form an active enzyme is restricted to a single mammalian family member, Ago2. This conclusion is supported both by biochemical analysis and by genetic studies in mutant MEFs. (2) Single amino acid substitutions within Ago2 that convert residues to those present in closely related proteins negate RISC cleavage. (3) The structure of the P. furiosis Argonaute protein reveals provocative structural similarities between the PIWI domain and the RNase H domains, providing a hypothesis for the method by which Argonaute cleaves its substrates. This hypothesis was tested by introducing mutations in the predicted Ago2 active site. It is extremely unlikely that such mutations could affect interactions with other proteins, because they are buried within a cleft of Ago (Liu, 2004).
These studies indicate that the Argonaute proteins that are unable to form cleavage-competent RISC differ from Ago2 at key positions that do not include the putative metal-coordinating residues themselves. However, it is not yet possible, based either on biochemical or structural studies, to provide a precise explanation for the catalytic defects in these proteins. It is conceivable that Ago1 and Ago3 fail to coordinate the catalytic metal or that the structure of the active site is distorted sufficiently that a bound metal is unable to access the scissile phosphate. Alternatively, catalytic mechanisms with two metal ions have been proposed for RNase H, which leaves open the possibility that catalytically inert Ago family members might lack structures essential to bind the second metal ion (Liu, 2004).
The relationship between the nuclease domain in PIWI and conserved nuclease domains in viral reverse transcriptases, transposases, and viral integrases has potential evolutionary implications. In Drosophila, plants, and C. elegans, the RNAi pathway has a major role in controlling parasitic nucleic acids such as viruses and transposons. The fact that the RNAi machinery shares a core structural domain with viruses and transposons suggests that this nucleic acid immune system may have arisen in part by pirating components from the replication and movement machineries of the very elements that RNAi protects against. This hypothesis is made even more poignant by considering the role of RNA-dependent RNA polymerases in RNAi, their functional relationship to viral replicases, and the possibility that the siRNAs themselves might first have served as primers that enable such replicases to duplicate primordial genomes (Liu, 2004).
RNA interference is implemented through the action of the RNA-induced silencing complex (RISC). Although Argonaute2 has been identified as the catalytic center of RISC, the RISC polypeptide composition and assembly using short interfering RNA (siRNA) duplexes has remained elusive. RISC is shown to be composed of Dicer, the double-stranded RNA binding protein TRBP, and Argonaute2. This complex can cleave target RNA using precursor microRNA (pre-miRNA) hairpin as the source of siRNA. Although RISC can also utilize duplex siRNA, it displays a nearly 10-fold greater activity using the pre-miRNA Dicer substrate. RISC distinguishes the guide strand of the siRNA from the passenger strand and specifically incorporates the guide strand. Importantly, ATP is not required for miRNA processing, RISC assembly, or multiple rounds of target-RNA cleavage. These results define the composition of RISC and demonstrate that miRNA processing and target-RNA cleavage are coupled (Gregory 2005).
This study shows that, although RISC could utilize the 22 nt duplex as the source of the siRNA, it displays far greater activity once a pre-miRNA, a substrate of Dicer, is used as the source of siRNA. These results strongly support the contention that Dicer cleavage activity is tightly coupled into the effector step of RNAi mediated by Ago2. The coupling of the two enzymatic activities makes ample biological sense since, once the duplex RNA is cleaved by Dicer, it could be unwound and handed over to Ago2 for target-RNA cleavage in a concerted reaction. The data showing a physical and functional coupling of pre-miRNA processing and RISC assembly also provide a mechanistic framework that explains the observations that 27 nt double-stranded RNAs or short hairpin RNAs, both of which are Dicer substrates, are considerably more potent triggers of RNAi than the short duplex siRNA. This study shows that, although RISC can utilize the 22 nt duplex as the source of the siRNA, it displays far greater activity once a pre-miRNA, a substrate of Dicer, is used as the source of siRNA. These results strongly support the contention that Dicer cleavage activity is tightly coupled into the effector step of RNAi mediated by Ago2. The coupling of the two enzymatic activities makes ample biological sense since, once the duplex RNA is cleaved by Dicer, it could be unwound and handed over to Ago2 for target-RNA cleavage in a concerted reaction. The data showing a physical and functional coupling of pre-miRNA processing and RISC assembly also provide a mechanistic framework that explains the observations that 27 nt double-stranded RNAs or short hairpin RNAs, both of which are Dicer substrates, are considerably more potent triggers of RNAi than the short duplex siRNA (Gregory, 2005)
Small interfering RNAs (siRNAs) are the mediators of mRNA degradation in the process of RNA interference (RNAi). A human biochemical system is described that recapitulates siRNA-mediated target RNA degradation. By using affinity-tagged siRNAs, it has been demonstrated that a single-stranded siRNA resides in the RNA-induced silencing complex (RISC) together with eIF2C1 and/or eIF2C2 (human GERp95) Argonaute proteins. RISC is rapidly formed in HeLa cell cytoplasmic extract supplemented with 21 nt siRNA duplexes, but also by adding single-stranded antisense RNAs, which range in size between 19 and 29 nucleotides. Single-stranded antisense siRNAs are also effectively silencing genes in HeLa cells, especially when 5'-phosphorylated, and expand the repertoire of RNA reagents suitable for gene targeting (Martinez, 2002).
It has been proposed that siRNAs act as primers for target RNA-templated dsRNA synthesis, even though homologs of the RNA-dependent RNA polymerases known to participate in gene silencing in other systems are apparently not encoded in D. melanogaster or mammalian genomes. Analysis of the fate of siRNA duplexes in the HeLa cell system does not provide evidence for such a siRNA-primed activity, but indicates that the predominant pathway for siRNA-mediated gene silencing is sequence-specific endonucleolytic target RNA degradation. Further evidence against siRNA-induced propagation of gene silencing in mammalian systems is that (1) the silenced gene returns to normal levels between 5 to 9 days posttransfection; (2) simultaneously expressed isoforms can be selectively targeted by siRNA duplexes (Martinez, 2002 and references therein).
RNA interference (RNAi) is an important means of eliminating mRNAs, but the intracellular location of RNA-induced silencing complex (RISC) remains unknown. Argonaute 2, a key component of RISC, is not randomly distributed but concentrates in mRNA decay centers that are known as cytoplasmic bodies. The localization of Argonaute 2 in decay centers is not altered by the presence or absence of small interfering RNAs or their targeted mRNAs. However, RNA is required for the integrity of cytoplasmic bodies because RNase eliminates Argonaute 2 localization. In addition, Argonaute 1, another Argonaute family member, is concentrated in cytoplasmic bodies. These results provide new insight into the mechanism of RNAi function (Sen, 2005).
MicroRNAs (miRNAs) are approximately 21-nucleotide-long RNA molecules regulating gene expression in multicellular eukaryotes. In metazoa, miRNAs act by imperfectly base-pairing with the 3' untranslated region of target messenger RNAs (mRNAs) and repressing protein accumulation by an unknown mechanism. Endogenous let-7 microribonucleoproteins (miRNPs) or the tethering of Argonaute (Ago) proteins to reporter mRNAs in human cells inhibit translation initiation. M(7)G-cap-independent translation is not subject to repression, suggesting that miRNPs interfere with recognition of the cap. Repressed mRNAs, Ago proteins, and miRNAs were all found to accumulate in processing bodies. It is proposed that localization of mRNAs to these structures is a consequence of translational repression (Pillai, 2005).
microRNAs (miRNAs) bind to Argonaute (Ago) proteins and inhibit translation or promote degradation of mRNA targets. Human let-7 miRNA inhibits translation initiation of mRNA targets in an m7G cap-dependent manner and also appears to block protein production, but the molecular mechanism(s) involved is unknown and the role of Ago proteins in translational regulation remains elusive. This study identified a motif (MC) within the Mid domain of Ago proteins, which bears significant similarity to the m7G cap-binding domain of eIF4E, an essential translation initiation factor. Conserved aromatic residues were identified within the MC motif of human Ago2 that are required for binding to the m7G cap and for translational repression but do not affect the assembly of Ago2 with miRNA or its catalytic activity. It is proposed that Ago2 represses the initiation of mRNA translation by binding to the m7G cap of mRNA targets, thus likely precluding the recruitment of eIF4E (Kiriakidou, 2007).
An important feature of miRNA-directed translational repression is its apparent cooperativity: increasing the number of miRNA recognition elements (MREs) in the 3′-UTR of an mRNA target enhances translational repression. Cooperativity is also seen when multiple MREs for different miRNAs are found in the 3′-UTR of the same mRNA target, arguing that common factors, notably Ago proteins, bound to all miRNAs are responsible for the enhanced translational repression. Indeed, this cooperativity is accurately recapitulated in experiments with tethered Ago2; increasing the number of BoxB sites in the 3′-UTR of the reporter leads to enhancement of the translational repression by λN-HA-Ago2. It is proposed that multiple MREs, within the same mRNA target, increase the number of Ago2 molecules bound to the mRNA, thus increasing the probability that they will interact with the m7G cap and augment translational repression by limiting availability of the m7G cap to eIF4E. In this model, Ago2 binds to m7G cap less efficiently than eIF4E. Therefore, optimal repression by Ago2 and thus optimal eIF4E competition would require multiple Ago2 molecules. Weak Ago2 binding to the m7G cap also makes biological sense, since an Ago2 protein with high affinity to the m7G cap would lead to generalized and strong translational inhibition. This model is also consistent with weak translational repression of mRNA targets that bear single MREs. Indeed, the vast majority of mRNA targets contain a single MRE for any given miRNA and the level of translational repression is typically modest (usually 1.5- to 2-fold repression). Such modest and noncomplete repression may also explain why many miRNAs cosediment with actively translating, endogenous, mRNAs in polysomes. Lastly, these findings do not exclude additional mechanisms of miRNA and Ago regulation, perhaps in the presence of additional factors such as inhibition of protein production on actively translating ribosomes during elongation or degradation of mRNAs (Kiriakidou, 2007).
An important observation is that the MC motif is not detected in Ago proteins from organisms that do not contain miRNAs, or do not use miRNAs for translational repression. Specifically, all mammalian Ago proteins and certain Ago proteins from nematodes and flies, where translational repression by miRNAs has been demonstrated, contain the MC domain, and thus these Ago proteins may be capable of repressing translation. The MC domain is present in Drosophila AGO1, which is required for miRNA function, but not in Drosophila AGO2, which functions predominantly in siRNA pathways, although more recent studies show overlapping functions of Ago1 and Ago2 pathways in flies. The MC domain is present in C.elegans ALG-1 and ALG-2 Ago proteins but absent from the remaining 25 members of the C.elegans Argonaute protein family, consistent with the finding that there are distinct RNAi-related pathways in nematodes, with ALG-1 and ALG-2 proteins participating in the microRNA pathway and all other nematode Argonaute proteins being associated with exo- or endo-RNAi pathways. Finally, the MC domain is absent from Ago proteins in organisms that do not have miRNAs such as fission yeast and Archaea. Although the MC motif is not found in Archaeal Agos, the structures of the P. furiosus and A. aeolicus Ago proteins show that a major portion of the Mid domain is accessible and thus may be capable of interacting with other factors. The MC domain is also not present in PIWI proteins, which are almost exclusively expressed in the germline. Notably, tethering of HIWI, a human PIWI protein, in the 3'-UTR of RL-5BoxB, is unable to repress RL translation. In contrast, tethering of all human Ago proteins (Ago1-4) in the 3'-UTR of RL-5BoxB results in strong repression of RL translation. These studies along with the finding that translational repression is unaffected in Ago2 null mouse embryonic fibroblasts also show that the endonuclease activity of mammalian Ago proteins is not required for translational repression. In flies, PIWI proteins associate with repeat-associated siRNAs. Mammalian PIWI proteins do not assemble with miRNAs or siRNAs but bind to slightly larger RNAs termed piRNAs. The mouse MIWI protein can associate with m7GTP sepharose, suggesting that MIWI proteins may also function in translation. However, since the MC domain is absent from the MIWI protein, it is possible that MIWI contains another cap-binding motif or associates with the cap-analog resin indirectly, via interactions with another cap-binding protein. However, the biochemical function of MIWI proteins and of piRNAs is unknown, and it is difficult to ascertain the functional consequences of this interaction at this point. Finally, the absence of the MC motif from plant Agos is intriguing and suggests that plant miRNAs may not be capable of repressing translation through interactions with the cap (but other mechanisms cannot be excluded). So far translational repression by miRNAs in plants has only been implicated for the control of very few mRNA targets, while most known plant miRNAs show extensive complementarity with their targets, directing target mRNA cleavage (Kiriakidou, 2007).
The RNA-Induced Silencing Complex (RISC) is a ribonucleoprotein particle composed of a single-stranded short interfering RNA (siRNA) and an endonucleolytically active Argonaute protein, capable of cleaving mRNAs complementary to the siRNA. The mechanism by which RISC cleaves a target RNA is well understood, however it remains enigmatic how RISC finds its target RNA. This study shows, both in vitro and in vivo, that the accessibility of the target site correlates directly with the efficiency of cleavage, demonstrating that RISC is unable to unfold structured RNA. In the course of target recognition, RISC transiently contacts single-stranded RNA nonspecifically and promotes siRNA-target RNA annealing. Furthermore, the 5' part of the siRNA within RISC creates a thermodynamic threshold that determines the stable association of RISC and the target RNA. RISC exhibits annealing activity which promotes siRNA-target RNA interactions and therefore renders nucleic-acid hybridization more efficient compared to the annealing of a single-stranded siRNA to a target in the absence of hAgo2. This activity might rely on (1) the siRNA-organization, with a helical geometry of the 5'-region favoring duplex formation, or (2) a nonspecific affinity of RISC toward RNA substrates, that might relate to the overall basicity of hAgo proteins. This study therefore provide mechanistic insights by revealing features of RISC and target RNAs that are crucial to achieve efficiency and specificity in RNA interference (Ameres, 2007).
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