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In eukaryotes, small RNA guides, such as small interfering RNAs and microRNAs, direct AGO-clade Argonaute proteins to regulate gene expression and defend the genome against external threats. Only animals make a second clade of Argonaute proteins: PIWI proteins. PIWI proteins use PIWI-interacting RNAs (piRNAs) to repress complementary transposon transcripts 1 , 2 . In theory, transposons could evade silencing through target site mutations that reduce piRNA complementarity. Here we report that, unlike AGO proteins, PIWI proteins efficiently cleave transcripts that are only partially paired to their piRNA guides. Examination of target binding and cleavage by mouse and sponge PIWI proteins revealed that PIWI slicing tolerates mismatches to any target nucleotide, including those flanking the scissile phosphate. Even canonical seed pairing is dispensable for PIWI binding or cleavage, unlike plant and animal AGOs, which require uninterrupted target pairing from the seed to the nucleotides past the scissile bond 3 , 4 . PIWI proteins are therefore better equipped than AGO proteins to target newly acquired or rapidly diverging endogenous transposons without recourse to new small RNA guides. Conversely, the minimum requirements for PIWI slicing are sufficient to avoid inadvertent silencing of host RNAs. Our results demonstrate the biological advantage of PIWI over AGO proteins in defending the genome against transposons and suggest an explanation for why the piRNA pathway was retained in animal evolution. Of the two types of Argonaute proteins produced by animals, AGO and PIWI, PIWI proteins can bind RNAs with less complementarity, enabling efficient silencing of transposons without the need to produce new RNA guides.

Although eukaryotic and long prokaryotic Argonaute proteins (pAgos) cleave nucleic acids, some short pAgos lack nuclease activity and hydrolyse NAD(P) + to induce bacterial cell death 1 . Here we present a hierarchical activation pathway for SPARTA, a short pAgo consisting of an Argonaute (Ago) protein and TIR–APAZ, an associated protein 2 . SPARTA progresses through distinct oligomeric forms, including a monomeric apo state, a monomeric RNA–DNA-bound state, two dimeric RNA–DNA-bound states and a tetrameric RNA–DNA-bound active state. These snapshots together identify oligomerization as a mechanistic principle of SPARTA activation. The RNA–DNA-binding channel of apo inactive SPARTA is occupied by an auto-inhibitory motif in TIR–APAZ. After the binding of RNA–DNA, SPARTA transitions from a monomer to a symmetric dimer and then an asymmetric dimer, in which two TIR domains interact through charge and shape complementarity. Next, two dimers assemble into a tetramer with a central TIR cluster responsible for hydrolysing NAD(P) + . In addition, we observe unique features of interactions between SPARTA and RNA–DNA, including competition between the DNA 3′ end and the auto-inhibitory motif, interactions between the RNA G2 nucleotide and Ago, and splaying of the RNA–DNA duplex by two loops exclusive to short pAgos. Together, our findings provide a mechanistic basis for the activation of short pAgos, a large section of the Ago superfamily. Cryo-electron microscopy structures and biochemical analyses provide insight into how short prokaryotic Argonaute proteins are assembled and activated, and reveal that oligomerization has a key role in driving catalytic activity.

Argonaute proteins use nucleic acid guides to find and bind specific DNA or RNA target sequences. Argonaute proteins have diverse biological functions and many retain their ancestral endoribonuclease activity, cleaving the phosphodiester bond between target nucleotides t10 and t11. In animals, the PIWI proteins—a specialized class of Argonaute proteins—use 21–35 nucleotide PIWI-interacting RNAs (piRNAs) to direct transposon silencing, protect the germline genome, and regulate gene expression during gametogenesis 1 . The piRNA pathway is required for fertility in one or both sexes of nearly all animals. Both piRNA production and function require RNA cleavage catalysed by PIWI proteins. Spermatogenesis in mice and other placental mammals requires three distinct, developmentally regulated PIWI proteins: MIWI (PIWIL1), MILI (PIWIL2) and MIWI2 2 – 4 (PIWIL4). The piRNA-guided endoribonuclease activities of MIWI and MILI are essential for the production of functional sperm 5 , 6 . piRNA-directed silencing in mice and insects also requires GTSF1, a PIWI-associated protein of unknown function 7 – 12 . Here we report that GTSF1 potentiates the weak, intrinsic, piRNA-directed RNA cleavage activities of PIWI proteins, transforming them into efficient endoribonucleases. GTSF1 is thus an example of an auxiliary protein that potentiates the catalytic activity of an Argonaute protein. The evolutionarily conserved RNA-binding protein GTSF1 and its homologues interact with members of the PIWI class of Argonaute proteins, increasing the efficiency of the RNA-cleaving activity of PIWI proteins, an essential function across the animal kingdom.

PIWI-clade Argonaute proteins associate with PIWI-interacting RNAs (piRNAs), and silence transposons in animal gonads. Here, we report the crystal structure of the Drosophila PIWI-clade Argonaute Piwi in complex with endogenous piRNAs, at 2.9 Å resolution. A structural comparison of Piwi with other Argonautes highlights the PIWI-specific structural features, such as the overall domain arrangement and metal-dependent piRNA recognition. Our structural and biochemical data reveal that, unlike other Argonautes including silkworm Siwi, Piwi has a non-canonical DVDK tetrad and lacks the RNA-guided RNA cleaving slicer activity. Furthermore, we find that the Piwi mutant with the canonical DEDH catalytic tetrad exhibits the slicer activity and readily dissociates from less complementary RNA targets after the slicer-mediated cleavage, suggesting that the slicer activity could compromise the Piwi-mediated co-transcriptional silencing. We thus propose that Piwi lost the slicer activity during evolution to serve as an RNA-guided RNA-binding platform, thereby ensuring faithful co-transcriptional silencing of transposons. PIWI-clade Argonautes bind PIWI-interacting RNAs (piRNAs) to silence transposons and maintain genome integrity in animal gonads. Here the authors present the crystal structure of a Drosophila Piwi–piRNA complex isolated from cultured fly ovarian somatic cells and find that it contains a non-canonical DVDK tetrad, and lacks slicer activity in vitro.

Background The renewed interest in epigenetics has led to the understanding that both the environment and individual lifestyle can directly interact with the epigenome to influence its dynamics. Epigenetic phenomena are mediated by DNA methylation, stable chromatin modifications and non-coding RNA-associated gene silencing involving specific proteins called epigenetic factors. Multiple organisms, ranging from plants to yeast and mammals, have been used as model systems to study epigenetics. The interactions between parasites and their hosts are models of choice to study these mechanisms because the selective pressures are strong and the evolution is fast. The asexually reproducing root-knot nematodes (RKN) offer different advantages to study the processes and mechanisms involved in epigenetic regulation. RKN genomes sequencing and annotation have identified numerous genes, however, which of those are involved in the adaption to an environment and potentially relevant to the evolution of plant-parasitism is yet to be discovered. Results Here, we used a functional comparative annotation strategy combining orthology data, mining of curated genomics as well as protein domain databases and phylogenetic reconstructions. Overall, we show that (i) neither RKN, nor the model nematode Caenorhabditis elegans possess any DNA methyltransferases (DNMT) (ii) RKN do not possess the complete machinery for DNA methylation on the 6th position of adenine (6mA) (iii) histone (de)acetylation and (de)methylation pathways are conserved between C. elegans and RKN, and the corresponding genes are amplified in asexually reproducing RKN (iv) some specific non-coding RNA families found in plant-parasitic nematodes are dissimilar from those in C. elegans . In the asexually reproducing RKN Meloidogyne incognita , expression data from various developmental stages supported the putative role of these proteins in epigenetic regulations. Conclusions Our results refine previous predictions on the epigenetic machinery of model species and constitute the most comprehensive description of epigenetic factors relevant to the plant-parasitic lifestyle and/or asexual mode of reproduction of RKN. Providing an atlas of epigenetic factors in RKN is an informative resource that will enable researchers to explore their potential role in adaptation of these parasites to their environment.

Background RNA interference (RNAi) leads to sequence specific knock-down of gene expression and has emerged as an important tool to analyse gene functions, pathway analysis and gene therapy. Although RNAi is a conserved cellular process involving common elements and factors, species-specific differences have been observed among different eukaryotes. Identification of components for RNAi pathway is pursued intensively and successful genome-wide screens have been performed for components of RNAi pathways in various organisms. Functional comparative genomics analysis offers evolutionary insight that forms basis of discoveries of novel RNAi-factors within related organisms. Keeping in view the academic and commercial utility of insect derived cell-line from Spodoptera frugiperda , we pursued the identification and functional analysis of components of RNAi-machinery of Sf21 cell-line using genome-wide application. Results The genome and transcriptome of Sf21 was assembled and annotated. In silico application of comparative genome analysis among insects allowed us to identify several RNAi factors in Sf21 line. The candidate RNAi factors from assembled genome were validated by knockdown analysis of candidate factors using the siRNA screens on the Sf21-gfp reporter cell-line. Forty two (42) potential factors were identified using the cell based assay. These include core RNAi elements including Dicer-2, Argonaute-1, Drosha, Aubergine and auxiliary modules like chromatin factors, RNA helicases, RNA processing module, signalling allied proteins and others. Phylogenetic analyses and domain architecture revealed that Spodoptera frugiperda homologs retained identity with Lepidoptera ( Bombyx mori ) or Coleoptera ( Tribolium castaneum ) sustaining an evolutionary conserved scaffold in post-transcriptional gene silencing paradigm within insects. Conclusion The database of RNAi-factors generated by whole genome association survey offers comprehensive outlook about conservation as well as specific differences of the proteins of RNAi machinery. Understanding the interior involved in different phases of gene silencing also offers impending tool for RNAi-based applications.

The plant protein ARGONAUTE1 (AGO1) functions in multiple RNA-silencing pathways, including those of microRNAs, key regulators of growth and development. Genetic analysis of ago1 mutants with informative defects has provided valuable insights into AGOl's biological functions. Tomato encodes two AGO1 homologs (SlAGO1s), but mutants have not been described to date. To analyze SlAGO1s' involvement in development, we confirmed that both undergo decay in the presence of the Polerovirus silencing suppressor P0 and produce a transgenic responder line (OP:P0HA) that, upon transactivation, expresses P0 C-terminally fused to a hemagglutinin (HA) tag (P0HA) and destabilizes SlAGO1s at the site of expression. By crossing OP:P0HA with a battery of driver lines, constitutive as well as organ- and stage-specific SlAGO1 downregulation was induced in the F1 progeny. Activated plants exhibited various developmental phenotypes that partially overlapped with those of Arabidopsis ago1 mutants. Plants that constitutively expressed P0HA had reduced SlAGO1 levels and increased accumulation of miRNA targets, indicating compromised SlAGO1-mediated silencing. Consistent with this, they exhibited pleiotropic morphological defects and their growth was arrested post-germination. Transactivation of P0HA in young leaf and floral organ primordia dramatically modified corresponding organ morphology, including the radialization of leaflets, petals and anthers, suggesting that SlAGO1s' activities are required for normal lateral organ development and polarity. Overall, our results suggest that the OP:P0HA responder line can serve as a valuable tool to suppress SlAGO1 silencing pathways in tomato. The suppression of additional SlAGOs by P0HA and its contribution to the observed phenotypes awaits investigation.

Background The sequencing of the genome of the pea aphid Acyrthosiphon pisum revealed an unusual expansion of the miRNA machinery, with two argonaute-1 , two dicer-1 and four pasha gene copies. In this report, we have undertaken a deeper evolutionary analysis of the phylogenetic timing of these gene duplications and of the associated selective pressures by sequencing the two copies of ago-1 and dcr-1 in different aphid species of the subfamily Aphidinae. We have also carried out an analysis of the expression of both copies of ago-1 and dcr-1 by semi-quantitative PCR in different morphs of the pea aphid life cycle. Results The analysis has shown that the duplication of ago-1 occurred in an ancestor of the subfamily Aphidinae while the duplication of dcr-1 appears to be more recent. Besides, it has confirmed a pattern of one conserved copy and one accelerated copy for both genes, and has revealed the action of positive selection on several regions of the fast-evolving ago-1b . On the other hand, the semi-quantitative PCR experiments have revealed a differential expression of these genes between the morphs of the parthenogenetic and the sexual phases of Acyrthosiphon pisum . Conclusions The discovery of these gene duplications in the miRNA machinery of aphids opens new perspectives of research about the regulation of gene expression in these insects. Accelerated evolution, positive selection and differential expression affecting some of the copies of these genes suggests the possibility of a neofunctionalization of these duplicates, which might play a role in the display of the striking phenotypic plasticity of aphids.

Background Bacteria and bacteriophages (phages) are locked in a coevolutionary “arms race” to outcompete one another with novel systems and strategies. Regularly outnumbered tenfold by phages, bacteria have responded to the constant threat of phage predation by evolving a vast array of sophisticated defense systems. Among these, prokaryotic Argonautes (pAgos) are nucleic acid-guided endonucleases that target complementary sequences of invading mobile genetic elements (MGEs). However, as the preference for targeting MGE sequences has been demonstrated in only a limited number of pAgos, their precise physiological functions remain elusive. Here, we discovered a pAgo in Exiguobacterium sp. AB2, EsAgo, encoded in close proximity to other putative defense systems on the E . AB2 genome. Such clustering into genomic “defense islands” is a common phenomenon among prokaryotic defense systems, further implicating pAgos with a role in host defense. Accordingly, we had sought to characterize EsAgo as a nucleic acid-guided nucleic acid-targeting nuclease against MGEs for bacterial defense in this study. Results Using sequence to structure homology tools, we show that the predicted model of EsAgo exhibits the structural characteristics typical of a full-length, catalytically active, DNA-guided pAgo. Akin to other pAgos, EsAgo uses a divalent cation cofactor to indiscriminately “chop” plasmids in vitro. Furthermore, a site-directed double mutant of EsAgo bearing two missense mutations at the catalytic site exhibited significantly reduced levels of this random plasmid-degrading activity. Lastly, when EsAgo was supplied with synthetic 5’-P ssDNA guides, random nuclease activity was attenuated. Conclusions These findings suggest that EsAgo functions as a DNA-interfering nuclease with or without DNA guides. Within the cell, it is possible that EsAgo utilizes this mechanism to screen and destroy foreign genetic elements. Moreover, the potential capacity for specific dsDNA cleavage at moderate temperatures gives rise to intriguing possibilities of repurposing EsAgo as a programmable nuclease for future biotechnological use.

MicroRNAs are small noncoding RNAs that play an important role in the regulation of various biological processes through their interaction with cellular messenger RNAs. They are frequently dysregulated in cancer and have shown great potential as tissue-based markers for cancer classification and prognostication. microRNAs are also present in extracellular human body fluids such as serum, plasma, saliva, and urine. Most of circulating microRNAs are present in human plasma and serum cofractionate with the Argonaute2 (Ago2) protein. However, circulating microRNAs have been also found in membrane-bound vesicles such as exosomes. Since microRNAs circulate in the bloodstream in a highly stable, extracellular form, they may be used as blood-based biomarkers for cancer and other diseases. A knowledge base of extracellular circulating miRNAs is a fundamental tool for biomedical research. In this work, we present miRandola, a comprehensive manually curated classification of extracellular circulating miRNAs. miRandola is connected to miRò, the miRNA knowledge base, allowing users to infer the potential biological functions of circulating miRNAs and their connections with phenotypes. The miRandola database contains 2132 entries, with 581 unique mature miRNAs and 21 types of samples. miRNAs are classified into four categories, based on their extracellular form: miRNA-Ago2 (173 entries), miRNA-exosome (856 entries), miRNA-HDL (20 entries) and miRNA-circulating (1083 entries). miRandola is available online at: http://atlas.dmi.unict.it/mirandola/index.html.