Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA.

Argonaute proteins use single-stranded RNA or DNA guides to target complementary nucleic acids. This allows eukaryotic Argonaute proteins to mediate RNA interference and long prokaryotic Argonaute proteins to interfere with invading nucleic acids. The function and mechanisms of the phylogenetically distinct short prokaryotic Argonaute proteins remain poorly understood. We demonstrate that short prokaryotic Argonaute and the associated TIR-APAZ (SPARTA) proteins form heterodimeric complexes. Upon guide RNA-mediated target DNA binding, four SPARTA heterodimers form oligomers in which TIR domain-mediated NAD(P)ase activity is unleashed. When expressed in Escherichia coli, SPARTA is activated in the presence of highly transcribed multicopy plasmid DNA, which causes cell death through NAD(P)+ depletion. This results in the removal of plasmid-invaded cells from bacterial cultures. Furthermore, we show that SPARTA can be repurposed for the programmable detection of DNA sequences. In conclusion, our work identifies SPARTA as a prokaryotic immune system that reduces cell viability upon RNA-guided detection of invading DNA.

The role of the Cer1 transposon in horizontal transfer of transgenerational memory.

Animals face both external and internal dangers: pathogens threaten from the environment, and unstable genomic elements threaten from within. C. elegans protects itself from pathogens by "reading" bacterial small RNAs, using this information to both induce avoidance and transmit memories for four generations. Here, we found that memories can be transferred from either lysed animals or from conditioned media to naive animals via Cer1 retrotransposon-encoded virus-like particles. Moreover, Cer1 functions internally at the step of transmission of information from the germline to neurons and is required for learned avoidance. The presence of the Cer1 retrotransposon in wild C. elegans strains correlates with the ability to learn and inherit small-RNA-induced pathogen avoidance. Together, these results suggest that C. elegans has co-opted a potentially dangerous retrotransposon to instead protect itself and its progeny from a common pathogen through its inter-tissue signaling ability, hijacking this genomic element for its own adaptive immunity benefit.

Neuronal Small RNAs Control Behavior Transgenerationally.

It is unknown whether the activity of the nervous system can be inherited. In Caenorhabditis elegans nematodes, parental responses can transmit heritable small RNAs that regulate gene expression transgenerationally. In this study, we show that a neuronal process can impact the next generations. Neurons-specific synthesis of RDE-4-dependent small RNAs regulates germline amplified endogenous small interfering RNAs (siRNAs) and germline gene expression for multiple generations. Further, the production of small RNAs in neurons controls the chemotaxis behavior of the progeny for at least three generations via the germline Argonaute HRDE-1. Among the targets of these small RNAs, we identified the conserved gene saeg-2, which is transgenerationally downregulated in the germline. Silencing of saeg-2 following neuronal small RNA biogenesis is required for chemotaxis under stress. Thus, we propose a small-RNA-based mechanism for communication of neuronal processes transgenerationally.

LTR-Retrotransposon Control by tRNA-Derived Small RNAs.

Transposon reactivation is an inherent danger in cells that lose epigenetic silencing during developmental reprogramming. In the mouse, long terminal repeat (LTR)-retrotransposons, or endogenous retroviruses (ERV), account for most novel insertions and are expressed in the absence of histone H3 lysine 9 trimethylation in preimplantation stem cells. We found abundant 18 nt tRNA-derived small RNA (tRF) in these cells and ubiquitously expressed 22 nt tRFs that include the 3' terminal CCA of mature tRNAs and target the tRNA primer binding site (PBS) essential for ERV reverse transcription. We show that the two most active ERV families, IAP and MusD/ETn, are major targets and are strongly inhibited by tRFs in retrotransposition assays. 22 nt tRFs post-transcriptionally silence coding-competent ERVs, while 18 nt tRFs specifically interfere with reverse transcription and retrotransposon mobility. The PBS offers a unique target to specifically inhibit LTR-retrotransposons, and tRF-targeting is a potentially highly conserved mechanism of small RNA-mediated transposon control.

Circular Concatemers of Ultra-Short DNA Segments Produce Regulatory RNAs.

In the ciliated protozoan Paramecium tetraurelia, Piwi-associated small RNAs are generated upon the elimination of tens of thousands of short transposon-derived DNA segments as part of development. These RNAs then target complementary DNA for elimination in a positive feedback process, contributing to germline defense and genome stability. In this work, we investigate the formation of these RNAs, which we show to be transcribed directly from the short (length mode 27 bp) excised DNA segments. Our data support a mechanism whereby the concatenation and circularization of excised DNA segments provides a template for RNA production. This process allows the generation of a double-stranded RNA for Dicer-like protein cleavage to give rise to a population of small regulatory RNAs that precisely match the excised DNA sequences. VIDEO ABSTRACT.

Crystal Structure of Silkworm PIWI-Clade Argonaute Siwi Bound to piRNA.

PIWI-clade Argonaute proteins associate with PIWI-interacting RNAs (piRNAs) and silence transposable elements in animal gonads. Here, we report the crystal structure of a silkworm PIWI-clade Argonaute, Siwi, bound to the endogenous piRNA, at 2.4 Å resolution. Siwi adopts a bilobed architecture consisting of N-PAZ and MID-PIWI lobes, in which the 5' and 3' ends of the bound piRNA are anchored by the MID-PIWI and PAZ domains, respectively. A structural comparison of Siwi with AGO-clade Argonautes reveals notable differences in their nucleic-acid-binding channels, likely reflecting the distinct lengths of their guide RNAs and their mechanistic differences in guide RNA loading and cleavage product release. In addition, the structure reveals that Siwi and prokaryotic, but not eukaryotic, AGO-clade Argonautes share unexpected similarities, such as metal-dependent 5'-phosphate recognition and a potential structural transition during the catalytic-tetrad formation. Overall, this study provides a critical starting point toward a mechanistic understanding of piRNA-mediated transposon silencing.

A Small RNA-Catalytic Argonaute Pathway Tunes Germline Transcript Levels to Ensure Embryonic Divisions.

Multiple division cycles without growth are a characteristic feature of early embryogenesis. The female germline loads proteins and RNAs into oocytes to support these divisions, which lack many quality control mechanisms operating in somatic cells undergoing growth. Here, we describe a small RNA-Argonaute pathway that ensures early embryonic divisions in C. elegans by employing catalytic slicing activity to broadly tune, instead of silence, germline gene expression. Misregulation of one target, a kinesin-13 microtubule depolymerase, underlies a major phenotype associated with pathway loss. Tuning of target transcript levels is guided by the density of homologous small RNAs, whose generation must ultimately be related to target sequence. Thus, the tuning action of a small RNA-catalytic Argonaute pathway generates oocytes capable of supporting embryogenesis. We speculate that the specialized nature of germline chromatin led to the emergence of small RNA-catalytic Argonaute pathways in the female germline as a post-transcriptional control layer to optimize oocyte composition.

Small RNAs and Hfq capture unfolded RNA target sites during transcription.

Small ribonucleoproteins (sRNPs) target nascent precursor RNAs to guide folding, modification, and splicing during transcription. Yet, rapid co-transcriptional folding of the RNA can mask sRNP sites, impeding target recognition and regulation. To examine how sRNPs target nascent RNAs, we monitored binding of bacterial Hfq⋅DsrA sRNPs to rpoS transcripts using single-molecule co-localization co-transcriptional assembly (smCoCoA). We show that Hfq⋅DsrA recursively samples the mRNA before transcription of the target site to poise it for base pairing with DsrA. We adapted smCoCoA to precisely measure when the target site is synthesized and revealed that Hfq⋅DsrA often binds the mRNA during target site synthesis close to RNA polymerase (RNAP). We suggest that targeting transcripts near RNAP allows an sRNP to capture a site before the transcript folds, providing a kinetic advantage over post-transcriptional targeting. We propose that other sRNPs may also use RNAP-proximal targeting to hasten recognition and regulation.

tRNA renovatio: Rebirth through fragmentation.

tRNA function is based on unique structures that enable mRNA decoding using anticodon trinucleotides. These structures interact with specific aminoacyl-tRNA synthetases and ribosomes using 3D shape and sequence signatures. Beyond translation, tRNAs serve as versatile signaling molecules interacting with other RNAs and proteins. Through evolutionary processes, tRNA fragmentation emerges as not merely random degradation but an act of recreation, generating specific shorter molecules called tRNA-derived small RNAs (tsRNAs). These tsRNAs exploit their linear sequences and newly arranged 3D structures for unexpected biological functions, epitomizing the tRNA "renovatio" (from Latin, meaning renewal, renovation, and rebirth). Emerging methods to uncover full tRNA/tsRNA sequences and modifications, combined with techniques to study RNA structures and to integrate AI-powered predictions, will enable comprehensive investigations of tRNA fragmentation products and new interaction potentials in relation to their biological functions. We anticipate that these directions will herald a new era for understanding biological complexity and advancing pharmaceutical engineering.

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.

Small RNAs guide histone methylation in Arabidopsis embryos.

Epigenetic reprogramming occurs during gametogenesis as well as during embryogenesis to reset the genome for early development. In flowering plants, many heterochromatic marks are maintained in sperm, but asymmetric DNA methylation is mostly lost. Asymmetric DNA methylation is dependent on small RNA but the re-establishment of silencing in embryo is not well understood. Here we demonstrate that small RNAs direct the histone H3 lysine 9 dimethylation during Arabidopsis thaliana embryonic development, together with asymmetric DNA methylation. This de novo silencing mechanism depends on the catalytic domain of SUVH9, a Su(Var)3-9 homolog thought to be catalytically inactive.

Noncanonical Roles of tRNAs: tRNA Fragments and Beyond.

As one of the most abundant and conserved RNA species, transfer RNAs (tRNAs) are well known for their role in reading the codons on messenger RNAs and translating them into proteins. In this review, we discuss the noncanonical functions of tRNAs. These include tRNAs as precursors to novel small RNA molecules derived from tRNAs, also called tRNA-derived fragments, that are abundant across species and have diverse functions in different biological processes, including regulating protein translation, Argonaute-dependent gene silencing, and more. Furthermore, the role of tRNAs in biosynthesis and other regulatory pathways, including nutrient sensing, splicing, transcription, retroelement regulation, immune response, and apoptosis, is reviewed. Genome organization and sequence variation of tRNA genes are also discussed in light of their noncanonical functions. Lastly, we discuss the recent applications of tRNAs in genome editing and microbiome sequencing.

Small RNAs, Large Networks: Posttranscriptional Regulons in Gram-Negative Bacteria.

Small regulatory RNA (sRNAs) are key mediators of posttranscriptional gene control in bacteria. Assisted by RNA-binding proteins, a single sRNA often modulates the expression of dozens of genes, and thus sRNAs frequently adopt central roles in regulatory networks. Posttranscriptional regulation by sRNAs comes with several unique features that cannot be achieved by transcriptional regulators. However, for optimal network performance, transcriptional and posttranscriptional control mechanisms typically go hand-in-hand. This view is reflected by the ever-growing class of mixed network motifs involving sRNAs and transcription factors, which are ubiquitous in biology and whose regulatory properties we are beginning to understand. In addition, sRNA activity can be antagonized by base-pairing with sponge RNAs, adding yet another layer of complexity to these networks. In this article, we summarize the regulatory concepts underlying sRNA-mediated gene control in bacteria and discuss how sRNAs shape the output of a network, focusing on several key examples.

The molecular basis of phenotypic evolution: beyond the usual suspects.

It has been well documented that mutations in coding DNA or cis-regulatory elements underlie natural phenotypic variation in many organisms. However, the development of sophisticated functional tools in recent years in a wide range of traditionally non-model systems have revealed many 'unusual suspects' in the molecular bases of phenotypic evolution, including upstream open reading frames (uORFs), cryptic splice sites, and small RNAs. Furthermore, large-scale genome sequencing, especially long-read sequencing, has identified a cornucopia of structural variation underlying phenotypic divergence and elucidated the composition of supergenes that control complex multi-trait polymorphisms. In this review article we highlight recent studies that demonstrate this great diversity of molecular mechanisms producing adaptive genetic variation and the panoply of evolutionary paths leading to the 'grandeur of life'.

A network of small RNAs regulates sporulation initiation in Clostridioides difficile.

The obligate anaerobic, enteric pathogen Clostridioides difficile persists in the intestinal tract by forming antibiotic-resistant endospores that contribute to relapsing and recurrent infections. Despite the importance of sporulation for C. difficile pathogenesis, environmental cues and molecular mechanisms that regulate sporulation initiation remain ill-defined. Here, by using RIL-seq to globally capture the Hfq-dependent RNA-RNA interactome, we discovered a network of small RNAs that bind to mRNAs encoding sporulation-related genes. We show that two of these small RNAs, SpoX and SpoY, regulate translation of the master regulator of sporulation, Spo0A, in an opposing manner, which ultimately leads to altered sporulation rates. Infection of antibiotic-treated mice with SpoX and SpoY deletion mutants revealed a global effect on gut colonization and intestinal sporulation. Our work uncovers an elaborate RNA-RNA interactome controlling the physiology and virulence of C. difficile and identifies a complex post-transcriptional layer in the regulation of spore formation in this important human pathogen.

Time-Resolved Small RNA Sequencing Unravels the Molecular Principles of MicroRNA Homeostasis.

Argonaute-bound microRNAs silence mRNA expression in a dynamic and regulated manner to control organismal development, physiology, and disease. We employed metabolic small RNA sequencing for a comprehensive view on intracellular microRNA kinetics in Drosophila. Based on absolute rate of biogenesis and decay, microRNAs rank among the fastest produced and longest-lived cellular transcripts, disposing up to 105 copies per cell at steady-state. Mature microRNAs are produced within minutes, revealing tight intracellular coupling of biogenesis that is selectively disrupted by pre-miRNA-uridylation. Control over Argonaute protein homeostasis generates a kinetic bottleneck that cooperates with non-coding RNA surveillance to ensure faithful microRNA loading. Finally, regulated small RNA decay enables the selective rapid turnover of Ago1-bound microRNAs, but not of Ago2-bound small interfering RNAs (siRNAs), reflecting key differences in the robustness of small RNA silencing pathways. Time-resolved small RNA sequencing opens new experimental avenues to deconvolute the timescales, molecular features, and regulation of small RNA silencing pathways in living cells.

Multidomain Convergence of Argonaute during RISC Assembly Correlates with the Formation of Internal Water Clusters.

Despite the relevance of Argonaute proteins in RNA silencing, little is known about the structural steps of small RNA loading to form RNA-induced silencing complexes (RISCs). We report the 1.9 Å crystal structure of human Argonaute4 with guide RNA. Comparison with the previously determined apo structure of Neurospora crassa QDE2 revealed that the PIWI domain has two subdomains. Binding of guide RNA fastens the subdomains, thereby rearranging the active-site residues and increasing the affinity for TNRC6 proteins. We also identified two water pockets beneath the nucleic acid-binding channel that appeared to stabilize the mature RISC. Indeed, mutating the water-pocket residues of Argonaute2 and Argonaute4 compromised RISC assembly. Simulations predict that internal water molecules are exchangeable with the bulk solvent but always occupy specific positions at the domain interfaces. These results suggest that after guide RNA-driven conformational changes, water-mediated hydrogen-bonding networks tie together the converged domains to complete the functional RISC structure.

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.

Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function.

Reproductive phasiRNAs (phased, small interfering RNAs) are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt (nucleotides) phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at the premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 (Dicer-like 5) for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely i) not triggered by microRNAs, ii) not loaded by AGO18 proteins, and iii) not capable of mediating PHAS precursor cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known.

The global RNA-RNA interactome of Klebsiella pneumoniae unveils a small RNA regulator of cell division.

The ubiquitous RNA chaperone Hfq is involved in the regulation of key biological processes in many species across the bacterial kingdom. In the opportunistic human pathogen Klebsiella pneumoniae, deletion of the hfq gene affects the global transcriptome, virulence, and stress resistance; however, the ligands of the major RNA-binding protein in this species have remained elusive. In this study, we have combined transcriptomic, co-immunoprecipitation, and global RNA interactome analyses to compile an inventory of conserved and species-specific RNAs bound by Hfq and to monitor Hfq-mediated RNA-RNA interactions. In addition to dozens of RNA-RNA pairs, our study revealed an Hfq-dependent small regulatory RNA (sRNA), DinR, which is processed from the 3' terminal portion of dinI mRNA. Transcription of dinI is controlled by the master regulator of the SOS response, LexA. As DinR accumulates in K. pneumoniae in response to DNA damage, the sRNA represses translation of the ftsZ transcript by occupation of the ribosome binding site. Ectopic overexpression of DinR causes depletion of ftsZ mRNA and inhibition of cell division, while deletion of dinR antagonizes cell elongation in the presence of DNA damage. Collectively, our work highlights the important role of RNA-based gene regulation in K. pneumoniae and uncovers the central role of DinR in LexA-controlled division inhibition during the SOS response.