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Mutations in Conserved Residues of the C. elegans microRNA Argonaute ALG-1 Identify Separable Functions in ALG-1 miRISC Loading and Target Repression  [PDF]
Anna Y. Zinovyeva,Samir Bouasker,Martin J. Simard,Christopher M. Hammell,Victor Ambros
PLOS Genetics , 2014, DOI: doi/10.1371/journal.pgen.1004286
Abstract: microRNAs function in diverse developmental and physiological processes by regulating target gene expression at the post-transcriptional level. ALG-1 is one of two Caenorhabditis elegans Argonautes (ALG-1 and ALG-2) that together are essential for microRNA biogenesis and function. Here, we report the identification of novel antimorphic (anti) alleles of ALG-1 as suppressors of lin-28(lf) precocious developmental phenotypes. The alg-1(anti) mutations broadly impair the function of many microRNAs and cause dosage-dependent phenotypes that are more severe than the complete loss of ALG-1. ALG-1(anti) mutant proteins are competent for promoting Dicer cleavage of microRNA precursors and for associating with and stabilizing microRNAs. However, our results suggest that ALG-1(anti) proteins may sequester microRNAs in immature and functionally deficient microRNA Induced Silencing Complexes (miRISCs), and hence compete with ALG-2 for access to functional microRNAs. Immunoprecipitation experiments show that ALG-1(anti) proteins display an increased association with Dicer and a decreased association with AIN-1/GW182. These findings suggest that alg-1(anti) mutations impair the ability of ALG-1 miRISC to execute a transition from Dicer-associated microRNA processing to AIN-1/GW182 associated effector function, and indicate an active role for ALG/Argonaute in mediating this transition.
Viral Protein Inhibits RISC Activity by Argonaute Binding through Conserved WG/GW Motifs  [PDF]
Ana Giner equal contributor,Lóránt Lakatos equal contributor ,Meritxell García-Chapa,Juan José López-Moya ,József Burgyán
PLOS Pathogens , 2010, DOI: 10.1371/journal.ppat.1000996
Abstract: RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as an antiviral mechanism in higher plants and insects. To overcome antiviral RNA silencing, viruses express silencing-suppressor proteins. These viral proteins can target one or more key points in the silencing machinery. Here we show that in Sweet potato mild mottle virus (SPMMV, type member of the Ipomovirus genus, family Potyviridae), the role of silencing suppressor is played by the P1 protein (the largest serine protease among all known potyvirids) despite the presence in its genome of an HC-Pro protein, which, in potyviruses, acts as the suppressor. Using in vivo studies we have demonstrated that SPMMV P1 inhibits si/miRNA-programmed RISC activity. Inhibition of RISC activity occurs by binding P1 to mature high molecular weight RISC, as we have shown by immunoprecipitation. Our results revealed that P1 targets Argonaute1 (AGO1), the catalytic unit of RISC, and that suppressor/binding activities are localized at the N-terminal half of P1. In this region three WG/GW motifs were found resembling the AGO-binding linear peptide motif conserved in metazoans and plants. Site-directed mutagenesis proved that these three motifs are absolutely required for both binding and suppression of AGO1 function. In contrast to other viral silencing suppressors analyzed so far P1 inhibits both existing and de novo formed AGO1 containing RISC complexes. Thus P1 represents a novel RNA silencing suppressor mechanism. The discovery of the molecular bases of P1 mediated silencing suppression may help to get better insight into the function and assembly of the poorly explored multiprotein containing RISC.
Convergent Transmission of RNAi Guide-Target Mismatch Information across Argonaute Internal Allosteric Network  [PDF]
Thomas T. Joseph,Roman Osman
PLOS Computational Biology , 2012, DOI: 10.1371/journal.pcbi.1002693
Abstract: In RNA interference, a guide strand derived from a short dsRNA such as a microRNA (miRNA) is loaded into Argonaute, the central protein in the RNA Induced Silencing Complex (RISC) that silences messenger RNAs on a sequence-specific basis. The positions of any mismatched base pairs in an miRNA determine which Argonaute subtype is used. Subsequently, the Argonaute-guide complex binds and silences complementary target mRNAs; certain Argonautes cleave the target. Mismatches between guide strand and the target mRNA decrease cleavage efficiency. Thus, loading and silencing both require that signals about the presence of a mismatched base pair are communicated from the mismatch site to effector sites. These effector sites include the active site, to prevent target cleavage; the binding groove, to modify nucleic acid binding affinity; and surface allosteric sites, to control recruitment of additional proteins to form the RISC. To examine how such signals may be propagated, we analyzed the network of internal allosteric pathways in Argonaute exhibited through correlations of residue-residue interactions. The emerging network can be described as a set of pathways emanating from the core of the protein near the active site, distributed into the bulk of the protein, and converging upon a distributed cluster of surface residues. Nucleotides in the guide strand “seed region” have a stronger relationship with the protein than other nucleotides, concordant with their importance in sequence selectivity. Finally, any of several seed region guide-target mismatches cause certain Argonaute residues to have modified correlations with the rest of the protein. This arises from the aggregation of relatively small interaction correlation changes distributed across a large subset of residues. These residues are in effector sites: the active site, binding groove, and surface, implying that direct functional consequences of guide-target mismatches are mediated through the cumulative effects of a large number of internal allosteric pathways.
Clarifying mammalian RISC assembly in vitro
Grace S Tan, Barry G Garchow, Xuhang Liu, David Metzler, Marianthi Kiriakidou
BMC Molecular Biology , 2011, DOI: 10.1186/1471-2199-12-19
Abstract: The use of pre-miRNAs as guides by Ago2 bypasses Dicer activity and complicates in vitro RISC reconstitution. In this work, we characterized Ago2:pre-miRNA complexes and identified RNAs that are targeted by miRNAs but not their corresponding pre-miRNAs. Using these target RNAs we were able to recapitulate in vitro pre-miRNA processing and canonical RISC loading, and define the minimal factors required for these processes.Our results indicate that Ago2 and Dicer are sufficient for processing and loading of miRNAs into RISC. Furthermore, our studies suggest that Ago2 binds primarily to the 5'- and alternatively, to the 3'-end of select pre-miRNAs.MicroRNAs (miRNAs) are small (~22 nucleotide) noncoding RNAs that associate with Argonaute proteins in ribonucleoprotein complexes (miRNPs or RISCs) [1-5]. MicroRNAs play diverse regulatory roles in development and physiological cellular functions [6-8]. In addition, miRNAs are involved in a wide spectrum of human diseases including cancer, cardiovascular and autoimmune inflammatory conditions [8-14].Post-transcriptional nuclear processing of primary miRNA transcripts (pri-miRNAs) by the RNAse III enzyme Drosha and its RNA-binding partner, DiGeorge syndrome Critical Region gene 8 protein (DGCR8) [15-18], generates ~ 65-75 nucleotide (nt) hairpin-structured miRNA precursors (pre-miRNAs). Pre-miRNAs are subsequently bound to the RAN-GTP protein Exportin-5 and are transported to the cytoplasm [19-22] where they undergo processing by the RNAse III enzyme Dicer into ~ 22 nt RNA duplexes [23,24]. TAR RNA binding protein (TRBP) facilitates Dicer processing of pre-miRNAs [25-28]. Ago proteins act as RNA chaperones facilitating unwinding of RNA duplexes and loading of single-stranded miRNAs into Ago complexes [29,30]. The mechanisms involved in miRNA strand selection in mammals are poorly understood.We previously reported that endogenous Ago2-pre-miRNA (pre-miRNPs) complexes are detected in cytoplasmic and nuclear extracts of human ce
Components and Assembly of RNA-induced Silencing Complex

SONG Xue-Mei,YAN Fei,DU Li-Xin,

遗传 , 2006,
Abstract: Degradation of homologous RNA in RNA interference is carried out by functional RNA-induced silencing complex(RISC).RISC contains Dicer,Argonaute proein,siRNA and other components.Researching structures and functions of these components is primary important for understanding assembly and functional mechanism of RISC,as well as the whole RNAi pathway.Recent research works showed that Dicer,containing RNaseIII domain,is responsible for production of siRNA at the beginning of RNAi,and guarantees the stability of RISC intermediate in assembly process.As the core component of RISC,Argonaute protein functions as slicer to cleave target RNA and offers the binding site of siRNA in RISC assembly,which are depended on PIWI domain and PAZ domain separately.Although there is only one strand of siRNA that is the guider of RISC,the double stranded structural character of siRNA is determinant of RNAi.Except those,there are still other components with unknown functions in RISC.The knowledge about RISC components and assembly now,is basis of a presumed RISC assembly model.
Accurate microRNA target prediction correlates with protein repression levels
Manolis Maragkakis, Panagiotis Alexiou, Giorgio L Papadopoulos, Martin Reczko, Theodore Dalamagas, George Giannopoulos, George Goumas, Evangelos Koukis, Kornilios Kourtis, Victor A Simossis, Praveen Sethupathy, Thanasis Vergoulis, Nectarios Koziris, Timos Sellis, Panagiotis Tsanakas, Artemis G Hatzigeorgiou
BMC Bioinformatics , 2009, DOI: 10.1186/1471-2105-10-295
Abstract: DIANA-microT 3.0 is an algorithm for microRNA target prediction which is based on several parameters calculated individually for each microRNA and combines conserved and non-conserved microRNA recognition elements into a final prediction score, which correlates with protein production fold change. Specifically, for each predicted interaction the program reports a signal to noise ratio and a precision score which can be used as an indication of the false positive rate of the prediction.Recently, several computational target prediction programs were benchmarked based on a set of microRNA target genes identified by the pSILAC method. In this assessment DIANA-microT 3.0 was found to achieve the highest precision among the most widely used microRNA target prediction programs reaching approximately 66%. The DIANA-microT 3.0 prediction results are available online in a user friendly web server at http://www.microrna.gr/microT webciteMicroRNAs (miRNAs) are short, endogenously expressed RNA molecules that regulate gene expression by binding directly and preferably to the 3' untranslated region (3'UTR) of protein coding genes [1]. Each miRNA is 19-24 nucleotides in length and is processed from a longer transcript which is referred to as the primary transcript (pri-miRNA). These transcripts are processed in the cell nucleus to short, 70-nucleotide stem-loop structures known as pre-miRNAs. Pre-miRNAs are processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer which cleaves the pre-miRNA stem-loop into two complementary short RNA molecules. One of these molecules is integrated into the RISC (RNA induced silencing complex) complex and guides the whole complex to the mRNA, thus inhibiting translation or inducing mRNA degradation [2]. Since their initial identification, miRNAs have been found to confer a novel layer of genetic regulation in a wide range of biological processes. miRNAs were first identified in 1993 [3] via classical genetic techniques
Up-Regulation of microRNA* Strands by Their Target Transcripts  [PDF]
Sung-Min Kang,Ji-Woong Choi,Su-Hyung Hong,Heon-Jin Lee
International Journal of Molecular Sciences , 2013, DOI: 10.3390/ijms140713231
Abstract: During microRNA (miRNA) biogenesis, one strand of a 21–23 nucleotide RNA duplex is preferentially selected for entry into an RNA-induced silencing complex (RISC). The other strand, known as the miRNA* species, is typically thought to be degraded. Previous studies have provided miRNA* selection models, but it remains unclear how the dominance of one arm arises during the biogenesis of miRNA. Using miRNA sponge-like methods, we cloned four tandem target sequences (artificial target) of miR-7b* and then measured miR-7b* expression levels after transfection of the artificial target. miR-7b* levels were found to significantly increase after transfection of the artificial target. We postulate that the abundance of target transcripts drives miRNA arm selection.
The evolution of core proteins involved in microRNA biogenesis
Dennis Murphy, Barry Dancis, James R Brown
BMC Evolutionary Biology , 2008, DOI: 10.1186/1471-2148-8-92
Abstract: Phylogenetic analyses show that all four miRNA pathway proteins were derived from large multiple protein families. As an example, vertebrate and invertebrate Argonaute (Ago) proteins diverged from a larger family of PIWI/Argonaute proteins found throughout eukaryotes. Further gene duplications among vertebrates after the evolution of chordates from urochordates but prior to the emergence of fishes lead to the evolution of four Ago paralogues. Invertebrate RISC RNA-binding proteins R2D2 and Loquacious are related to other RNA-binding protein families such as Staufens as well as vertebrate-specific TAR (HIV trans-activator RNA) RNA-binding protein (TRBP) and protein kinase R-activating protein (PACT). Export of small RNAs from the nucleus, including miRNA, is facilitated by three closely related karyopherin-related nuclear transporters, Exportin-5, Exportin-1 and Exportin-T. While all three exportins have direct orthologues in deutrostomes, missing exportins in arthropods (Exportin-T) and nematodes (Exportin-5) are likely compensated by dual specificities of one of the other exportin paralogues.Co-opting particular isoforms from large, diverse protein families seems to be a common theme in the evolution of miRNA biogenesis. Human miRNA biogenesis proteins have direct, orthologues in cold-blooded fishes and, in some cases, urochordates and deutrostomes. However, lineage specific expansions of Dicer in plants and invertebrates as well as Argonaute and RNA-binding proteins in vertebrates suggests that novel ncRNA regulatory mechanisms can evolve in relatively short evolutionary timeframes. The occurrence of multiple homologues to RNA-binding and Argonaute/PIWI proteins also suggests the possible existence of further pathways for additional types of ncRNAs.Recent studies have unveiled the critical roles that RNA interference (RNAi) mediated by small noncoding RNAs (ncRNAs) plays in the regulation of eukaryotic genes. One particular important ncRNA class is microRNA (miRNA
An endogenous F-box protein regulates ARGONAUTE1 in Arabidopsis thaliana
K Earley, MR Smith, R Weber, BD Gregory, RS Poethig
Silence , 2010, DOI: 10.1186/1758-907x-1-15
Abstract: Argonaute proteins are core components of the RNA-induced silencing complex (RISC) [1-3]. These proteins use microRNAs (miRNAs) and/or small interfering RNAs (siRNAs) as guides to direct RISC to a specific site in target mRNAs, resulting in the cleavage or translational repression of these target mRNAs. Some Argonaute proteins also promote transcriptional repression through their effect on chromatin structure [2-4].ARGONAUTE1 (AGO1) is one of 10 Argonaute proteins in Arabidopsis thaliana [2,5]. Genetic analyses [6-9], as well as the identification of the small RNAs that co-purify with AGO1 [10,11], indicate that AGO1 plays a central role in both miRNA and siRNA-mediated RNA silencing. Arabidopsis is exquisitely sensitive to the level of AGO1 activity, as evident from the broad range of phenotypes displayed by hypomorphic mutations of this gene [5,6,8,12]. In wild-type plants, the expression of AGO1 is maintained at a constant level by a negative feedback loop involving miR168. AGO1 is a target of miR168 and negatively regulates its own activity by promoting the activity and stability of miR168 [9,13] and by promoting the activity of siRNAs derived from the AGO1 transcript [14]. AGO1 activity is negatively regulated by PNH/ZLL/AGO10 [15] and positively regulated by SQUINT (SQN), the Arabidopsis orthologue of the protein chaperone, Cyclophilin-40 [12].Null alleles of SQN have a morphological phenotype that is nearly identical to the phenotype of weak loss-of-function alleles of AGO1 [12]. In order to identify genes involved in AGO1-mediated processes, we screened for mutations that suppress the phenotype of sqn-1. This screen yielded several alleles of the F-box gene FBW2. Here we show FBW2 is a negative regulator of AGO1 and controls the sensitivity of plants to the hormone abscisic acid.Previously we found that SQN directly or indirectly promotes AGO1 activity [12]. In particular, we showed that the phenotype of loss-of-function alleles of SQN can be largely, if not
microPIR: An Integrated Database of MicroRNA Target Sites within Human Promoter Sequences  [PDF]
Jittima Piriyapongsa, Chaiwat Bootchai, Chumpol Ngamphiw, Sissades Tongsima
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0033888
Abstract: Background microRNAs are generally understood to regulate gene expression through binding to target sequences within 3′-UTRs of mRNAs. Therefore, computational prediction of target sites is usually restricted to these gene regions. Recent experimental studies though have suggested that microRNAs may alternatively modulate gene expression by interacting with promoters. A database of potential microRNA target sites in promoters would stimulate research in this field leading to more understanding of complex microRNA regulatory mechanism. Methodology We developed a database hosting predicted microRNA target sites located within human promoter sequences and their associated genomic features, called microPIR (microRNA-Promoter Interaction Resource). microRNA seed sequences were used to identify perfect complementary matching sequences in the human promoters and the potential target sites were predicted using the RNAhybrid program. >15 million target sites were identified which are located within 5000 bp upstream of all human genes, on both sense and antisense strands. The experimentally confirmed argonaute (AGO) binding sites and EST expression data including the sequence conservation across vertebrate species of each predicted target are presented for researchers to appraise the quality of predicted target sites. The microPIR database integrates various annotated genomic sequence databases, e.g. repetitive elements, transcription factor binding sites, CpG islands, and SNPs, offering users the facility to extensively explore relationships among target sites and other genomic features. Furthermore, functional information of target genes including gene ontologies, KEGG pathways, and OMIM associations are provided. The built-in genome browser of microPIR provides a comprehensive view of multidimensional genomic data. Finally, microPIR incorporates a PCR primer design module to facilitate experimental validation. Conclusions The proposed microPIR database is a useful integrated resource of microRNA-promoter target interactions for experimental microRNA researchers and computational biologists to study the microRNA regulation through gene promoter. The database can be freely accessed from: http://www4a.biotec.or.th/micropir.
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