Oligomerization-mediated activation of a short prokaryotic Argonaute.

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 death1. Here we present a hierarchical activation pathway for SPARTA, a short pAgo consisting of an Argonaute (Ago) protein and TIR-APAZ, an associated protein2. 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.

Relaxed targeting rules help PIWI proteins silence transposons.

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 transcripts1,2. 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 bond3,4. 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.

GTSF1 accelerates target RNA cleavage by PIWI-clade Argonaute proteins.

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 gametogenesis1. 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 MIWI22-4 (PIWIL4). The piRNA-guided endoribonuclease activities of MIWI and MILI are essential for the production of functional sperm5,6. piRNA-directed silencing in mice and insects also requires GTSF1, a PIWI-associated protein of unknown function7-12. 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.

piRNAclusterDB 2.0: update and expansion of the piRNA cluster database.

PIWI-interacting RNAs (piRNAs) and their partnering PIWI proteins defend the animal germline against transposable elements and play a crucial role in fertility. Numerous studies in the past have uncovered many additional functions of the piRNA pathway, including gene regulation, anti-viral defense, and somatic transposon repression. Further, comparative analyses across phylogenetic groups showed that the PIWI/piRNA system evolves rapidly and exhibits great evolutionary plasticity. However, the presence of so-called piRNA clusters as the major source of piRNAs is common to nearly all metazoan species. These genomic piRNA-producing loci are highly divergent across taxa and critically influence piRNA populations in different evolutionary lineages. We launched the initial version of the piRNA cluster database to facilitate research on regulation and evolution of piRNA-producing loci across tissues und species. In recent years the amount of small RNA sequencing data that was generated and the abundance of species that were studied has grown rapidly. To keep up with this recent progress, we have released a major update for the piRNA cluster database (https://www.smallrnagroup.uni-mainz.de/piRNAclusterDB), expanding it from 12 to a total of 51 species with hundreds of new datasets, and revised its overall structure to enable easy navigation through this large amount of data.

Crystal structure of Drosophila Piwi.

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.

Genome-wide expert annotation of the epigenetic machinery of the plant-parasitic nematodes Meloidogyne spp., with a focus on the asexually reproducing species.

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.

Sequence- and structure-based analysis of proteins involved in miRNA biogenesis.

miRNA biogenesis is a multistage process for the generation of a mature miRNA and involves several different proteins. In this work, we have carried out both sequence- and structure-based analysis for crucial proteins involved in miRNA biogenesis, namely Dicer, Drosha, Argonaute (Ago), and Exportin-5 to understand evolution of these proteins in animal kingdom and also to identify key sequence and structural features that are determinants of their function. Our analysis reveals that in animals the miRNA biogenesis pathway first originated in molluscs. The phylogeny of Dicer and Ago indicated evolution through gene duplication followed by sequence divergence that resulted in functional divergence. Our detailed structural analysis also revealed that RIIIDb domains of Drosha and Dicer, share significant similarity in sequence, structure, and substrate-binding pocket. On the other hand, PAZ domains of Dicer and Ago show only conservation of the substrate-binding pockets in the catalytic sites despite significant divergence in sequence and overall structure. Based on a comparative structural analysis of all four human Ago proteins (hAgo1-4) and their known biochemical activity, we have also attempted to identify key residues in Ago2 which are responsible for the unique slicer activity of hAgo2 among all isoforms. We have identified six key residues in N domain of hAgo2, which are located far away from the catalytic pocket, but might be playing a major role in slicer activity of hAgo2 protein because of their involvement in mRNA binding.

The classification, structure and functioning of Ago proteins in Eukaryotes.

Ago proteins are members of the highly specialized and conserved Argonaute family, primarily responsible for regulation of gene expression. As a part of RNA-induced silencing complexes (RISCs) Ago proteins are responsible for binding a short RNA and cleavage/inhibition of translation of target mRNAs. Phosphorylation may work as the switch between those two functions, but the role of magnesium ion concentration is also taken into consideration. Recent reports indicate that Ago proteins can interact with an mRNA and cause inhibition of translation without the participation of a short RNA. As key elements in RNA interference processes, Ago proteins are an important and intensively exploited area of research. Furthermore, these proteins are involved in the repair of DNA double-strand breaks by homologous recombination, modifications of chromatin, and alternative splicing. Their role in the cell cycle and senescence is also being studied. In addition, Ago expression is tissue-specific, which potentially may be used for diagnostic purposes. Understanding the mechanisms of Ago functioning is therefore crucial for understanding many cellular processes. The following article presents a detailed description of the Ago proteins including their post-translational modifications, recent data and hypotheses concerning their interactions with short RNAs and mRNAs as well as the mechanisms of siRNA/miRNA sorting into individual members of the Ago subfamily, and their role in eukaryotic cells. The latest classification of Ago proteins within the Argonaute family based on evolutionary studies and their possible interactions with DNA are also described.

Functional dissection of a plant Argonaute.

RNA guided ribonuclease complexes play central role in RNA interference. Members of the evolutionarily conserved Argonaute protein family form the catalytic cores of these complexes. Unlike a number of other plant Argonautes, the role of AGO2 has been obscure until recently. Newer data, however, have indicated its involvement in various biotic and abiotic stress responses. Despite its suggested importance, there is no detailed characterization of this protein to date. Here we report cloning and molecular characterization of the AGO2 protein of the virological model plant Nicotiana benthamiana. We show that AGO2 can directly repress translation via various miRNA target site constellations (ORF, 3' UTR). Interestingly, although AGO2 seems to be able to silence gene expression in a slicing independent fashion, its catalytic activity is still a prerequisite for efficient translational repression. Additionally, mismatches between the 3' end of the miRNA guide strand and the 5' end of the target site enhance gene silencing by AGO2. Several functionally important amino acid residues of AGO2 have been identified that affect its small RNA loading, cleavage activity, translational repression potential and antiviral activity. The data presented here help us to understand how AGO2 aids plants to deal with stress.

Comparative functional characterization of the CSR-1 22G-RNA pathway in Caenorhabditis nematodes.

As a champion of small RNA research for two decades, Caenorhabditis elegans has revealed the essential Argonaute CSR-1 to play key nuclear roles in modulating chromatin, chromosome segregation and germline gene expression via 22G-small RNAs. Despite CSR-1 being preserved among diverse nematodes, the conservation and divergence in function of the targets of small RNA pathways remains poorly resolved. Here we apply comparative functional genomic analysis between C. elegans and Caenorhabditis briggsae to characterize the CSR-1 pathway, its targets and their evolution. C. briggsae CSR-1-associated small RNAs that we identified by immunoprecipitation-small RNA sequencing overlap with 22G-RNAs depleted in cbr-csr-1 RNAi-treated worms. By comparing 22G-RNAs and target genes between species, we defined a set of CSR-1 target genes with conserved germline expression, enrichment in operons and more slowly evolving coding sequences than other genes, along with a small group of evolutionarily labile targets. We demonstrate that the association of CSR-1 with chromatin is preserved, and show that depletion of cbr-csr-1 leads to chromosome segregation defects and embryonic lethality. This first comparative characterization of a small RNA pathway in Caenorhabditis establishes a conserved nuclear role for CSR-1 and highlights its key role in germline gene regulation across multiple animal species.

Genome wide screening of RNAi factors of Sf21 cells reveal several novel pathway associated proteins.

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.

Functional diversification of Argonautes in nematodes: an expanding universe.

In the last decade, many diverse RNAi (RNA interference) pathways have been discovered that mediate gene silencing at epigenetic, transcriptional and post-transcriptional levels. The diversity of RNAi pathways is inherently linked to the evolution of Ago (Argonaute) proteins, the central protein component of RISCs (RNA-induced silencing complexes). An increasing number of diverse Agos have been identified in different species. The functions of most of these proteins are not yet known, but they are generally assumed to play roles in development, genome stability and/or protection against viruses. Recent research in the nematode Caenorhabditis elegans has expanded the breadth of RNAi functions to include transgenerational epigenetic memory and, possibly, environmental sensing. These functions are inherently linked to the production of secondary siRNAs (small interfering RNAs) that bind to members of a clade of WAGOs (worm-specific Agos). In the present article, we review briefly what is known about the evolution and function of Ago proteins in eukaryotes, including the expansion of WAGOs in nematodes. We postulate that the rapid evolution of WAGOs enables the exceptional functional plasticity of nematodes, including their capacity for parasitism.

The developmental outcomes of P0-mediated ARGONAUTE destabilization in tomato.

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 AGO1'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.

Evolutionary study of duplications of the miRNA machinery in aphids associated with striking rate acceleration and changes in expression profiles.

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.