Thermus thermophilus Argonaute Functions in the Completion of DNA Replication.

In many eukaryotes, Argonaute proteins, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15- to 18-nt DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of gyrase and TtAgo activity slows growth and produces long sausage-like filaments in which the individual bacteria are linked by DNA. Finally, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication.

Single-Molecule Imaging Reveals that Argonaute Reshapes the Binding Properties of Its Nucleic Acid Guides.

Argonaute proteins repress gene expression and defend against foreign nucleic acids using short RNAs or DNAs to specify the correct target RNA or DNA sequence. We have developed single-molecule methods to analyze target binding and cleavage mediated by the Argonaute:guide complex, RISC. We find that both eukaryotic and prokaryotic Argonaute proteins reshape the fundamental properties of RNA:RNA, RNA:DNA, and DNA:DNA hybridization­a small RNA or DNA bound to Argonaute as a guide no longer follows the well-established rules by which oligonucleotides find, bind, and dissociate from complementary nucleic acid sequences. Argonautes distinguish substrates from targets with similar complementarity. Mouse AGO2, for example, binds tighter to miRNA targets than its RNAi cleavage product, even though the cleaved product contains more base pairs. By re-writing the rules for nucleic acid hybridization, Argonautes allow oligonucleotides to serve as specificity determinants with thermodynamic and kinetic properties more typical of RNA-binding proteins than of RNA or DNA.

High-throughput biochemical profiling reveals functional adaptation of a bacterial Argonaute.

Argonautes are nucleic acid-guided proteins that perform numerous cellular functions across all domains of life. Little is known about how distinct evolutionary pressures have shaped each Argonaute's biophysical properties. We applied high-throughput biochemistry to characterize how Thermus thermophilus Argonaute (TtAgo), a DNA-guided DNA endonuclease, finds, binds, and cleaves its targets. We found that TtAgo uses biophysical adaptations similar to those of eukaryotic Argonautes for rapid association but requires more extensive complementarity to achieve high-affinity target binding. Using these data, we constructed models for TtAgo association rates and equilibrium binding affinities that estimate the nucleic acid- and protein-mediated components of the target interaction energies. Finally, we showed that TtAgo cleavage rates vary widely based on the DNA guide, suggesting that only a subset of guides cleaves targets on physiologically relevant timescales.

Structural basis of microRNA biogenesis by Dicer-1 and its partner protein Loqs-PB.

In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM structures of Drosophila Dicer-1 that show how Dicer-1 and its partner Loqs­PB cooperate (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, and (4) following complete dicing and initial product release. Our reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer­1⋅Loqs­PB heterodimer. The Dicer-1 dsRBD and three Loqs­PB dsRBDs form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-miRNA cleavage shifts the dsRBDs and partially closes Dicer-1, which may promote product release. Our data suggest a model for how the Dicer­1⋅Loqs­PB complex affects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and product release.

The HP1 homolog rhino anchors a nuclear complex that suppresses piRNA precursor splicing.

piRNAs guide an adaptive genome defense system that silences transposons during germline development. The Drosophila HP1 homolog Rhino is required for germline piRNA production. We show that Rhino binds specifically to the heterochromatic clusters that produce piRNA precursors, and that binding directly correlates with piRNA production. Rhino colocalizes to germline nuclear foci with Rai1/DXO-related protein Cuff and the DEAD box protein UAP56, which are also required for germline piRNA production. RNA sequencing indicates that most cluster transcripts are not spliced and that rhino, cuff, and uap56 mutations increase expression of spliced cluster transcripts over 100-fold. LacI::Rhino fusion protein binding suppresses splicing of a reporter transgene and is sufficient to trigger piRNA production from a trans combination of sense and antisense reporters. We therefore propose that Rhino anchors a nuclear complex that suppresses cluster transcript splicing and speculate that stalled splicing differentiates piRNA precursors from mRNAs.

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.

To Degrade a MicroRNA, Destroy Its Argonaute Protein.

Han et al. (2020) and Shi et al. (2020) report that the E3 ubiquitin ligase ZSWIM8 senses when an RNA and an Argonaute protein-bound microRNA are extensively base paired and directs Argonaute destruction by the proteasome. The result is degradation of the microRNA.

Terminal modification, sequence, length, and PIWI-protein identity determine piRNA stability.

In animals, PIWI-interacting RNAs (piRNAs) silence transposons, fight viral infections, and regulate gene expression. piRNA biogenesis concludes with 3' terminal trimming and 2'-O-methylation. Both trimming and methylation influence piRNA stability. Our biochemical data show that multiple mechanisms destabilize unmethylated mouse piRNAs, depending on whether the piRNA 5' or 3' sequence is complementary to a trigger RNA. Unlike target-directed degradation of microRNAs, complementarity-dependent destabilization of piRNAs in mice and flies is blocked by 3' terminal 2'-O-methylation and does not require base pairing to both the piRNA seed and the 3' sequence. In flies, 2'-O-methylation also protects small interfering RNAs (siRNAs) from complementarity-dependent destruction. By contrast, pre-piRNA trimming protects mouse piRNAs from a degradation pathway unaffected by trigger complementarity. In testis lysate and in vivo, internal or 3' terminal uridine- or guanine-rich tracts accelerate pre-piRNA decay. Loss of both trimming and 2'-O-methylation causes the mouse piRNA pathway to collapse, demonstrating that these modifications collaborate to stabilize piRNAs.

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.

Argonaute divides its RNA guide into domains with distinct functions and RNA-binding properties.

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) guide Argonaute proteins to silence mRNA expression. Argonaute binding alters the properties of an RNA guide, creating functional domains. We show that the domains established by Argonaute-the anchor, seed, central, 3' supplementary, and tail regions-have distinct biochemical properties that explain the differences between how animal miRNAs and siRNAs bind their targets. Extensive complementarity between an siRNA and its target slows the rate at which fly Argonaute2 (Ago2) binds to and dissociates from the target. Highlighting its role in antiviral defense, fly Ago2 dissociates so slowly from extensively complementary target RNAs that essentially every fully paired target is cleaved. Conversely, mouse AGO2, which mainly mediates miRNA-directed repression, dissociates rapidly and with similar rates for fully paired and seed-matched targets. Our data narrow the range of biochemically reasonable models for how Argonaute-bound siRNAs and miRNAs find, bind, and regulate their targets.

Dicer partner proteins tune the length of mature miRNAs in flies and mammals.

Drosophila Dicer-1 produces microRNAs (miRNAs) from pre-miRNA, whereas Dicer-2 generates small interfering RNAs (siRNAs) from long dsRNA. Alternative splicing of the loquacious (loqs) mRNA generates three distinct Dicer partner proteins. To understand the function of each, we constructed flies expressing Loqs-PA, Loqs-PB, or Loqs-PD. Loqs-PD promotes both endo- and exo-siRNA production by Dicer-2. Loqs-PA or Loqs-PB is required for viability, but the proteins are not fully redundant: a specific subset of miRNAs requires Loqs-PB. Surprisingly, Loqs-PB tunes where Dicer-1 cleaves pre-miR-307a, generating a longer miRNA isoform with a distinct seed sequence and target specificity. The longer form of miR-307a represses glycerol kinase and taranis mRNA expression. The mammalian Dicer-partner TRBP, a Loqs-PB homolog, similarly tunes where Dicer cleaves pre-miR-132. Thus, Dicer-binding partner proteins change the choice of cleavage site by Dicer, producing miRNAs with target specificities different from those made by Dicer alone or Dicer bound to alternative protein partners.

UAP56 couples piRNA clusters to the perinuclear transposon silencing machinery.

piRNAs silence transposons during germline development. In Drosophila, transcripts from heterochromatic clusters are processed into primary piRNAs in the perinuclear nuage. The nuclear DEAD box protein UAP56 has been previously implicated in mRNA splicing and export, whereas the DEAD box protein Vasa has an established role in piRNA production and localizes to nuage with the piRNA binding PIWI proteins Ago3 and Aub. We show that UAP56 colocalizes with the cluster-associated HP1 variant Rhino, that nuage granules containing Vasa localize directly across the nuclear envelope from cluster foci containing UAP56 and Rhino, and that cluster transcripts immunoprecipitate with both Vasa and UAP56. Significantly, a charge-substitution mutation that alters a conserved surface residue in UAP56 disrupts colocalization with Rhino, germline piRNA production, transposon silencing, and perinuclear localization of Vasa. We therefore propose that UAP56 and Vasa function in a piRNA-processing compartment that spans the nuclear envelope.

The RNA-Binding ATPase, Armitage, Couples piRNA Amplification in Nuage to Phased piRNA Production on Mitochondria.

PIWI-interacting RNAs (piRNAs) silence transposons in Drosophila ovaries, ensuring female fertility. Two coupled pathways generate germline piRNAs: the ping-pong cycle, in which the PIWI proteins Aubergine and Ago3 increase the abundance of pre-existing piRNAs, and the phased piRNA pathway, which generates strings of tail-to-head piRNAs, one after another. Proteins acting in the ping-pong cycle localize to nuage, whereas phased piRNA production requires Zucchini, an endonuclease on the mitochondrial surface. Here, we report that Armitage (Armi), an RNA-binding ATPase localized to both nuage and mitochondria, links the ping-pong cycle to the phased piRNA pathway. Mutations that block phased piRNA production deplete Armi from nuage. Armi ATPase mutants cannot support phased piRNA production and inappropriately bind mRNA instead of piRNA precursors. We propose that Armi shuttles between nuage and mitochondria, feeding precursor piRNAs generated by Ago3 cleavage into the Zucchini-dependent production of Aubergine- and Piwi-bound piRNAs on the mitochondrial surface.

Maelstrom Represses Canonical Polymerase II Transcription within Bi-directional piRNA Clusters in Drosophila melanogaster.

In Drosophila, 23-30 nt long PIWI-interacting RNAs (piRNAs) direct the protein Piwi to silence germline transposon transcription. Most germline piRNAs derive from dual-strand piRNA clusters, heterochromatic transposon graveyards that are transcribed from both genomic strands. These piRNA sources are marked by the heterochromatin protein 1 homolog Rhino (Rhi), which facilitates their promoter-independent transcription, suppresses splicing, and inhibits transcriptional termination. Here, we report that the protein Maelstrom (Mael) represses canonical, promoter-dependent transcription in dual-strand clusters, allowing Rhi to initiate piRNA precursor transcription. Mael also represses promoter-dependent transcription at sites outside clusters. At some loci, Mael repression requires the piRNA pathway, while at others, piRNAs play no role. We propose that by repressing canonical transcription of individual transposon mRNAs, Mael helps Rhi drive non-canonical transcription of piRNA precursors without generating mRNAs encoding transposon proteins.

High-Throughput Analysis Reveals Rules for Target RNA Binding and Cleavage by AGO2.

Argonaute proteins loaded with microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC), which represses target RNA expression. Predicting the biological targets, specificity, and efficiency of both miRNAs and siRNAs has been hamstrung by an incomplete understanding of the sequence determinants of RISC binding and cleavage. We applied high-throughput methods to measure the association kinetics, equilibrium binding energies, and single-turnover cleavage rates of mouse AGO2 RISC. We find that RISC readily tolerates insertions of up to 7 nt in its target opposite the central region of the guide. Our data uncover specific guide:target mismatches that enhance the rate of target cleavage, suggesting novel siRNA design strategies. Using these data, we derive quantitative models for RISC binding and target cleavage and show that our in vitro measurements and models predict knockdown in an engineered cellular system.

Adaptation to P element transposon invasion in Drosophila melanogaster.

Transposons evolve rapidly and can mobilize and trigger genetic instability. Piwi-interacting RNAs (piRNAs) silence these genome pathogens, but it is unclear how the piRNA pathway adapts to invasion of new transposons. In Drosophila, piRNAs are encoded by heterochromatic clusters and maternally deposited in the embryo. Paternally inherited P element transposons thus escape silencing and trigger a hybrid sterility syndrome termed P-M hybrid dysgenesis. We show that P-M hybrid dysgenesis activates both P elements and resident transposons and disrupts the piRNA biogenesis machinery. As dysgenic hybrids age, however, fertility is restored, P elements are silenced, and P element piRNAs are produced de novo. In addition, the piRNA biogenesis machinery assembles, and resident elements are silenced. Significantly, resident transposons insert into piRNA clusters, and these new insertions are transmitted to progeny, produce novel piRNAs, and are associated with reduced transposition. P element invasion thus triggers heritable changes in genome structure that appear to enhance transposon silencing.

A Single Mechanism of Biogenesis, Initiated and Directed by PIWI Proteins, Explains piRNA Production in Most Animals.

In animals, PIWI-interacting RNAs (piRNAs) guide PIWI proteins to silence transposons and regulate gene expression. The mechanisms for making piRNAs have been proposed to differ among cell types, tissues, and animals. Our data instead suggest a single model that explains piRNA production in most animals. piRNAs initiate piRNA production by guiding PIWI proteins to slice precursor transcripts. Next, PIWI proteins direct the stepwise fragmentation of the sliced precursor transcripts, yielding tail-to-head strings of phased precursor piRNAs (pre-piRNAs). Our analyses detect evidence for this piRNA biogenesis strategy across an evolutionarily broad range of animals, including humans. Thus, PIWI proteins initiate and sustain piRNA biogenesis by the same mechanism in species whose last common ancestor predates the branching of most animal lineages. The unified model places PIWI-clade Argonautes at the center of piRNA biology and suggests that the ancestral animal-the Urmetazoan-used PIWI proteins both to generate piRNA guides and to execute piRNA function.