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Background: eNOS (endothelial nitric oxide synthase) is an endothelial cell (EC)–specific gene predominantly expressed in medium- to large-sized arteries where ECs experience atheroprotective laminar flow with high shear stress. Disturbed flow with lower average shear stress decreases eNOS transcription, which leads to the development of atherosclerosis, especially at bifurcations and curvatures of arteries. This prototypic arterial EC gene contains 2 distinct flow-responsive cis-DNA elements in the promoter, the shear stress response element (SSRE) and the KLF (Krüppel-like factor) element. Previous in vitro studies suggested their positive regulatory functions on flow-induced transcription of EC genes including eNOS. However, the in vivo function of these cis-DNA elements remains unknown. Methods: Insertional transgenic mice with a mutation at each flow-responsive cis-DNA element were generated using a murine eNOS promoter–β-galactosidase reporter by linker-scanning mutagenesis and compared with episomal-based mutations in vitro. DNA methylation at the eNOS proximal promoter in mouse ECs was assessed by bisulfite sequencing or pyrosequencing. Results: Wild type mice with a functional eNOS promoter–reporter transgene exhibited reduced endothelial reporter expression in the atheroprone regions of disturbed flow (n=5). It is surprising that the SSRE mutation abrogated reporter expression in ECs and was associated with aberrant hypermethylation at the eNOS proximal promoter (n=7). Reporter gene silencing was independent of transgene copy number and integration position, indicating that the SSRE is a critical cis-element necessary for eNOS transcription in vivo. The KLF mutation demonstrated an integration site–specific decrease in eNOS transcription, again with marked promoter methylation (n=8), suggesting that the SSRE alone is not sufficient for eNOS transcription in vivo. In wild type mice, the native eNOS promoter was significantly hypermethylated in ECs from the atheroprone regions where eNOS expression was markedly repressed by chronic disturbed flow, demonstrating that eNOS expression is regulated by flow-dependent DNA methylation that is region-specific in the arterial endothelium in vivo. Conclusions: We report, for the first time, that the SSRE and KLF elements are critical flow sensors necessary for a transcriptionally permissive, hypomethylated eNOS promoter in ECs under chronic shear stress in vivo. Moreover, eNOS expression is regulated by flow-dependent epigenetic mechanisms, which offers novel mechanistic insight on eNOS gene regulation in atherogenesis.

Mutations in the piRNA pathway protein components lead to transposon activation and fertility defects. In contrast, Gebert et al., (2021) saw no defects in transposon silencing or fertility when they deleted three large germline piRNA clusters in D. melanogaster.

piRNAs are small non-coding RNAs that guide the silencing of transposons and other targets in animal gonads. In Drosophila female germline, many piRNA source loci dubbed “piRNA clusters” lack hallmarks of active genes and exploit an alternative path for transcription, which relies on the Rhino-Deadlock-Cutoff (RDC) complex. RDC was thought to be absent in testis, so it remains to date unknown how piRNA cluster transcription is regulated in the male germline. We found that components of RDC complex are expressed in male germ cells during early spermatogenesis, from germline stem cells (GSCs) to early spermatocytes. RDC is essential for expression of dual-strand piRNA clusters and transposon silencing in testis; however, it is dispensable for expression of Y-linked Suppressor of Stellate piRNAs and therefore Stellate silencing. Despite intact Stellate repression, males lacking RDC exhibited compromised fertility accompanied by germline DNA damage and GSC loss. Thus, piRNA-guided repression is essential for normal spermatogenesis beyond Stellate silencing. While RDC associates with multiple piRNA clusters in GSCs and early spermatogonia, its localization changes in later stages as RDC concentrates on a single X-linked locus, AT-chX. Dynamic RDC localization is paralleled by changes in piRNA cluster expression, indicating that RDC executes a fluid piRNA program during different stages of spermatogenesis. These results disprove the common belief that RDC is dispensable for piRNA biogenesis in testis and uncover the unexpected, sexually dimorphic and dynamic behavior of a core piRNA pathway machinery., Author summary Large fractions of eukaryotic genomes are occupied by mobile genetic elements called transposons. Active transposons can move in the genome causing DNA damage and mutations, while inactive copies can contribute to chromosome organization and regulation of gene expression. Host cells employ several mechanisms to discriminate transposons from other genes and repress transposon activities. In germ cells, a conserved class of short RNAs called Piwi-interacting (pi)RNAs recognize target RNAs in both the nucleus and cytoplasm and then guide transposon repression by preventing their transcription and destroying their RNAs. piRNAs are encoded in extended genomic regions dubbed piRNA clusters. Previously, composition and regulation of piRNA clusters were studied in the female germline of fruit flies, where a nuclear protein complex, the RDC complex, was shown to promote non-canonical transcription of these regions. However, RDC was believed to be dispensable in males. Here, we showed that RDC is essential for transposon repression in males, and males lacking RDC exhibit compromised fertility and loss of germ cells. We found that RDC binds multiple piRNA clusters in early germ cells but concentrates on a single locus at later stages. Our results indicate dynamic regulation of loci that produce piRNAs and, therefore, piRNA targets throughout spermatogenesis.

PIWI proteins use guide piRNAs to repress selfish genomic elements, protecting the genomic integrity of gametes and ensuring the fertility of animal species. Efficient transposon repression depends on amplification of piRNA guides in the ping-pong cycle, which in Drosophila entails tight cooperation between two PIWI proteins, Aub and Ago3. Here we show that post-translational modification, symmetric dimethylarginine (sDMA), of Aub is essential for piRNA biogenesis, transposon silencing and fertility. Methylation is triggered by loading of a piRNA guide into Aub, which exposes its unstructured N-terminal region to the PRMT5 methylosome complex. Thus, sDMA modification is a signal that Aub is loaded with piRNA guide. Amplification of piRNA in the ping-pong cycle requires assembly of a tertiary complex scaffolded by Krimper, which simultaneously binds the N-terminal regions of Aub and Ago3. To promote generation of new piRNA, Krimper uses its two Tudor domains to bind Aub and Ago3 in opposite modification and piRNA-loading states. Our results reveal that post-translational modifications in unstructured regions of PIWI proteins and their binding by Tudor domains that are capable of discriminating between modification states is essential for piRNA biogenesis and silencing., PIWI protein contains arginine rich motifs that are post-translationally modified to symmetrically methylated arginine (sDMA) residues. Here the authors show that piRNA loading into Aub triggers sDMA modification which is recognized by Krimper to promote formation of Krimper-Aub-Ago3 complex for piRNA amplification in Drosophila.

Small noncoding piRNAs act as sequence-specific guides to repress complementary targets in Metazoa. Prior studies in Drosophila ovaries have demonstrated the function of the piRNA pathway in transposon silencing and therefore genome defense. However, the ability of the piRNA program to respond to different transposon landscapes and the role of piRNAs in regulating host gene expression remain poorly understood. Here, we comprehensively analyzed piRNA expression and defined the repertoire of their targets in Drosophila melanogaster testes. Comparison of piRNA programs between sexes revealed sexual dimorphism in piRNA programs that parallel sex-specific transposon expression. Using a novel bioinformatic pipeline, we identified new piRNA clusters and established complex satellites as dual-strand piRNA clusters. While sharing most piRNA clusters, the two sexes employ them differentially to combat the sex-specific transposon landscape. We found two piRNA clusters that produce piRNAs antisense to four host genes in testis, including CG12717/pirate, a SUMO protease gene. piRNAs encoded on the Y chromosome silence pirate, but not its paralog, to exert sex- and paralog-specific gene regulation. Interestingly, pirate is targeted by endogenous siRNAs in a sibling species, Drosophila mauritiana, suggesting distinct but related silencing strategies invented in recent evolution to regulate a conserved protein-coding gene.

Microbes occupy a major part of the planets’ living material; the greater part of the microorganisms including bacteria, viruses, spores, and fungi, is hazardous to living organisms including human beings. Infectious diseases kill over 17 million people per year according to WHO reports. Unfortunately, mortality rates of few years show new records due to the novel microbes including SARS, MERS, and COVID-19. Generally, numerous disease-causing bacteria produce poisonous chemicals that harm cells. Viruses aren’t made out of cells; however, they are smaller than cells. Recently, various researches have been conducted in the development for the protection against various pathogens, which is more essential in paving way to the discovery of antimicrobial agents. This chapter provides the overall information on the microbes and their protection including the mechanism behind the microbes. The antimicrobial textiles for hygienic and medical purposes are discussed comprehensively.

The novel coronavirus disease (COVID-19), has become the most critical global health challenge in recent history. With SARS-CoV-2 infection, there was an unexpectedly high and specific prevalence of olfactory and taste disorders (OTDs). These high rates of hyposmia and hypogeusia, initially reported as up to 89% in European case series, led to the global inclusion of loss of taste and/or smell as a distinctive feature of COVID-19. However, there is emerging evidence that there are striking differences in the rates of OTDs in East Asian countries where the disease first emerged, as compared to Western countries (15.8 vs. 60.9%, p-value < 0.01). This may be driven by either variations in SARS-CoV-2 subtypes presenting to different global populations or genotypic differences in hosts which alter the predisposition of these different populations to the neuroinvasiveness of SARS-CoV-2. We also found that rates of OTDs were significantly higher in objective testing for OTDs as compared to subjective testing (73.6 vs. 60.8%, p-value = 0.03), which is the methodology employed by most studies. Concurrently, it has also become evident that racial minorities across geographically disparate world populations suffer from disproportionately higher rates of COVID-19 infection and mortality. In this mini review, we aim to delineate and explore the varying rates of olfactory and taste disorders amongst COVID-19 patients, by focusing on their underlying geographical, testing, ethnic and socioeconomic differences. We examine the current literature for evidence of differences in the olfactory and gustatory manifestations of COVID-19 and discuss current pathophysiological hypotheses for such differences.

Argonaute proteins are programmable nucleases that are found in both eukaryotes and prokaryotes and provide defense against invading genetic elements. Although some prokaryotic Argonautes (pAgos) were shown to recognize RNA targets in vitro, the majority of studied pAgos have strict specificity toward DNA, which limits their practical use in RNA-centric applications. Here, we describe a unique KmAgo nuclease from the mesophilic bacterium Kurthia massiliensis that can be programmed with either DNA or RNA guides and can precisely cleave both DNA and RNA targets. KmAgo preferentially binds 16-20 nt long 5′-phosphorylated guide molecules with no strict specificity for their sequence and is active in a wide range of temperatures. In bacterial cells, KmAgo is loaded with small DNAs with no obvious sequence preferences suggesting that it can uniformly target genomic sequences. Target cleavage by KmAgo depends on the formation of secondary structure indicating that KmAgo can be used for structural probing of RNA targets. Mismatches between the guide and target sequences greatly affect the efficiency and precision of target cleavage, depending on the mismatch position and the nature of the reacting nucleic acid. These properties of KmAgo open the way for its use for highly specific nucleic acid detection and cleavage.

Recognition and repression of RNA targets by Argonaute proteins guided by small RNAs is the essence of RNA interference in eukaryotes. Argonaute proteins with diverse structures are also found in many bacterial and archaeal genomes. Recent studies revealed that, similarly to their eukaryotic counterparts, prokaryotic Argonautes (pAgos) may function in cell defense against foreign genetic elements but, in contrast, preferably act on DNA targets. Many crucial details of the pAgo action, and the roles of a plethora of pAgos with non-conventional architecture remain unknown. Here, we review available structural and biochemical data on pAgos and discuss their possible functions in host defense and other genetic processes in prokaryotic cells., In this review, Aravin and colleagues examine bacterial and archaeal Argonaute proteins, discuss their diverse architectures and their possible roles in host defense, proposing additional functions for Argonaute proteins in prokaryotic cells.

One of the main conditions of the species splitting from a common precursor lineage is the prevention of a gene flow between diverging populations. The study ofDrosophilainterspecific hybrids allows to reconstruct the speciation mechanisms and to identify hybrid incompatibility factors that maintain post-zygotic reproductive isolation between closely related species. The regulation, evolution, and maintenance of the testis-specificSte-Su(Ste)genetic system inDrosophila melanogasteris the subject of investigation worldwide. X-linked tandem testis-specificStellategenes encode proteins homologous to the regulatory β-subunit of protein kinase CK2, but they are permanently repressed in wild-type flies by the piRNA pathway via piRNAs originating from the homologous Y-linkedSu(Ste)locus. Derepression ofStellategenes caused bySu(Ste)piRNA biogenesis disruption leads to the accumulation of crystalline aggregates in spermatocytes, meiotic defects and male sterility. In this review we summarize current data about the origin, organization, evolution of theSte-Su(Ste)system, and piRNA-dependent regulation ofStellateexpression. TheSte-Su(Ste)system is fixed only in theD. melanogastergenome. According to our hypothesis, the acquisition of theSte-Su(Ste)system by a part of the ancient fly population appears to be the causative factor of hybrid sterility in crosses of female flies with males that do not carry Y-linkedSu(Ste)repeats. To support this scenario, we have directly demonstratedStellatederepression and the corresponding meiotic disorders in the testes of interspecies hybrids betweenD. melanogasterandD. mauritiana. This finding embraces our hypothesis about the contribution of theSte-Su(Ste)system and the piRNA pathway to the emergence of reproductive isolation ofD. melanogasterlineage from initial species.

In contrast to eukaryotic Argonaute proteins that act on RNA targets, prokaryotic Argonautes (pAgos) can target DNA, using either small RNA or small DNA guides for its recognition. Since pAgos can recognize only a single strand of DNA and lack a helicase activity, it remains unknown how double-stranded DNA can be bound both in vitro and in vivo. Here, using in vitro reconstitution and footprinting assays we analyze formation of specific complexes with target DNA by a catalytically inactive pAgo, RsAgo from Rhodobacter sphaeroides programmed with small guide RNAs. We showed that RsAgo can recognize a specific site in double-stranded DNA after stepwise reconstitution of the complex from individual oligonucleotides or after prior melting of the DNA target. When bound, RsAgo stabilizes an open DNA bubble corresponding to the length of the guide molecule and protects the target DNA from nuclease cleavage. The results suggest that RsAgo and, possibly, other RNA-guided pAgos cannot directly attack double-stranded DNA and likely require DNA opening by other cellular processes for their action.

Ribosomal RNAs (rRNAs) are essential components of the ribosome and are among the most abundant macromolecules in the cell. To ensure high rRNA level, eukaryotic genomes contain dozens to hundreds of rDNA genes, however, only a fraction of the rRNA genes seems to be active, while others are transcriptionally silent. We found that individual rDNA genes have high level of cell-to-cell heterogeneity in their expression inDrosophila melanogaster. Insertion of heterologous sequences into rDNA leads to repression associated with reduced expression in individual cells and decreased number of cells expressing rDNA with insertions. We found that SUMO (Small Ubiquitin-like Modifier) and SUMO ligase Ubc9 are required for efficient repression of interrupted rDNA units and variable expression of intact rDNA. Disruption of the SUMO pathway abolishes discrimination of interrupted and intact rDNAs and removes cell-to-cell heterogeneity leading to uniformly high expression of individual rDNA in single cells. Our results suggest that the SUMO pathway is responsible for both repression of interrupted units and control of intact rDNA expression.

SUMMARYpiRNAs are small non-coding RNAs that guide the silencing of transposons and other targets in animal gonads. InDrosophilafemale germline, many piRNA source loci dubbed ‘piRNA clusters’ lack hallmarks of active genes and exploit an alternative path for transcription, which relies on the Rhino-Deadlock-Cutoff (RDC) complex. It remains to date unknown how piRNA cluster transcription is regulated in the male germline. We found that components of RDC complex are expressed in male germ cells during early spermatogenesis, from germline stem cells (GSCs) to early spermatocytes. RDC is essential for expression of dual-strand piRNA clusters and transposon silencing in testis; however, it is dispensable for expression of Y-linkedSuppressor of StellatepiRNAs and thereforeStellatesilencing. Despite intactStellaterepression,rhimutant males exhibited compromised fertility accompanied by germline DNA damage and GSC loss. Thus, piRNA-guided repression is essential for normal spermatogenesis beyondStellatesilencing. While RDC associates with multiple piRNA clusters in GSCs and early spermatogonia, its localization changes in later stages as RDC concentrates on a single X-linked locus,AT-chX. Dynamic RDC localization is paralleled by changes in piRNA cluster expression, indicating that RDC executes a fluid piRNA program during different stages of spermatogenesis.

SUMMARYSmall non-coding piRNAs act as sequence-specific guides to repress complementary targets in Metazoa. Prior studies inDrosophilaovaries have demonstrated the function of piRNA pathway in transposon silencing and therefore genome defense. However, the ability of piRNA program to respond to different transposon landscape and the role of piRNAs in regulating host gene expression remain poorly understood. Here, we comprehensively analyzed piRNA expression and defined the repertoire of their targets inDrosophila melanogastertestes. Comparison of piRNA programs between sexes revealed sexual dimorphism in piRNA programs that parallel sex-specific transposon expression. Using a novel bioinformatic pipeline, we identified new piRNA clusters and established complex satellites as dual-strand piRNA clusters. While sharing most piRNA clusters, two sexes employ them differentially to combat sex-specific transposon landscape. We found several host genes targeted by piRNAs in testis, includingCG12717/pita, a SUMO protease gene. piRNAs encoded on Y chromosome silencepita, but not its paralog, to exert sex- and paralog-specific gene regulation. Interestingly,pitais targeted by endogenous siRNAs in a sibling species,Drosophila mauritiana, suggesting distinct but related silencing strategies invented in recent evolution to regulate a conserved protein-encoding gene.

Ribosomal RNAs (rRNAs) are essential components of the ribosome and are among the most abundant macromolecules in the cell. To ensure high rRNA level, eukaryotic genomes contain dozens to hundreds of rDNA genes, however, only a fraction of the rRNA genes seems to be active, while others are transcriptionally silent. We found that individual rDNA genes have high level of cell-to-cell heterogeneity in their expression inDrosophila melanogaster. Insertion of heterologous sequences into rDNA leads to repression associated with reduced expression in individual cells and decreased number of cells expressing rDNA with insertions. We found that SUMO (Small Ubiquitin-like Modifier) and SUMO ligase Ubc9 are required for efficient repression of interrupted rDNA units and variable expression of intact rDNA. Disruption of the SUMO pathway abolishes discrimination of interrupted and intact rDNAs and removes cell-to-cell heterogeneity leading to uniformly high expression of individual rDNA in single cells. Our results suggest that the SUMO pathway is responsible for both repression of interrupted units and control of intact rDNA expression.

Members of the conserved Argonaute protein family use small RNA guides to locate their mRNA targets and regulate gene expression and suppress mobile genetic elements in eukaryotes1,2. Argonautes are also present in many bacterial and archaeal species3–5. Unlike eukaryotic proteins, several prokaryotic Argonaute proteins use small DNA guides to cleave DNA, a process known as DNA interference6–10. However, the natural functions and targets of DNA interference are poorly understood, and the mechanisms of DNA guide generation and target discrimination remain unknown. Here we analyse the activity of a bacterial Argonaute nuclease from Clostridium butyricum (CbAgo) in vivo. We show that CbAgo targets multicopy genetic elements and suppresses the propagation of plasmids and infection by phages. CbAgo induces DNA interference between homologous sequences and triggers DNA degradation at double-strand breaks in the target DNA. The loading of CbAgo with locus-specific small DNA guides depends on both its intrinsic endonuclease activity and the cellular double-strand break repair machinery. A similar interaction was reported for the acquisition of new spacers during CRISPR adaptation, and prokaryotic genomes that encode Ago nucleases are enriched in CRISPR–Cas systems. These results identify molecular mechanisms that generate guides for DNA interference and suggest that the recognition of foreign nucleic acids by prokaryotic defence systems involves common principles. Argonaute protein from the bacterium C. butyricum targets multicopy genetic elements and functions in the suppression of plasmid and phage propagation, and there appears to be a DNA-mediated immunity pathway in prokaryotes.

Members of the conserved Argonaute protein family use small RNA guides to find their mRNA targets to regulate gene expression and suppress mobile genetic elements in eukaryotes. Argonautes are also present in many bacterial and archaeal species 1–3. Unlike eukaryotic proteins, several studied prokaryotic Argonautes use small DNA guides to cleave DNA, a process dubbed DNA interference 4–8. However, the natural functions and targets of DNA interference are poorly understood and the mechanisms of DNA guide generation and target discrimination remain unknown. Here, we studied the in vivo activities of a bacterial Argonaute nuclease CbAgo and demonstrated that it induces cleavage of multicopy genetic elements, including plasmids, transposons and repetitive chromosomal loci. Generation of small DNA guides employed by CbAgo requires cooperation between its intrinsic endonuclease activity and the cellular double-strand break repair machinery. The mechanism of guide generation ensures that small DNA guides are enriched in sequences that target foreign DNA and endows CbAgo with capacity to eliminate plasmids and fight phage infection. Similar principles may underlie the specificity of self-nonself discrimination by diverse defense systems in prokaryotes.

Members of the conserved Argonaute (Ago) protein family provide defence against invading nucleic acids in eukaryotes in the process of RNA interference. Many prokaryotes also contain Ago proteins that are predicted to be active nucleases; however, their functional activities in host cells remain poorly understood. Here, we characterize the in vitro and in vivo properties of the SeAgo protein from the mesophilic cyanobacterium Synechococcus elongatus. We show that SeAgo is a DNA-guided nuclease preferentially acting on single-stranded DNA targets, with non-specific guide-independent activity observed for double-stranded substrates. The SeAgo gene is steadily expressed in S. elongatus; however, its deletion or overexpression does not change the kinetics of cell growth. When purified from its host cells or from heterologous E. coli, SeAgo is loaded with small guide DNAs whose formation depends on the endonuclease activity of the argonaute protein. SeAgo co-purifies with SSB proteins suggesting that they may also be involved in DNA processing. The SeAgo-associated small DNAs are derived from diverse genomic locations, with certain enrichment for the proposed sites of chromosomal replication initiation and termination, but show no preference for an endogenous plasmid. Therefore, promiscuous genome sampling by SeAgo does not have great effects on cell physiology and plasmid maintenance.

International audience; Transgenerational epigenetic inheritance (TEI) describes the transmission of alternative functional states through multiple generations in the presence of the same genomic DNA sequence. Very little is known about the principles and the molecular mechanisms governing this type of inheritance. Here, by transiently enhancing 3D chromatin interactions, we established stable and isogenic Drosophila epilines that carry alternative epialleles, as defined by differential levels of Polycomb-dependent trimethylation of histone H3 Lys27 (forming H3K27me3). After being established, epialleles can be dominantly transmitted to naive flies and can induce paramutation. Importantly, epilines can be reset to a naive state by disruption of chromatin interactions. Finally, we found that environmental changes modulate the expressivity of the epialleles, and we extended our paradigm to naturally occurring phenotypes. Our work sheds light on how nuclear organization and Polycomb group (PcG) proteins contribute to epigenetically inheritable phenotypic variability.

Pachytene Piwi-interacting RNAs (piRNAs) are abundant small non-coding RNAs expressed in mammalian germ lines. A new study indicates that, among the diverse pool of piRNA sequences, a small number act as highly selective guides that induce cleavage of coding and non-coding transcripts, thus promoting piRNA generation and regulating gene expression.

Histone 3 lysine 9 trimethylation (H3K9me3) is a conserved histone modification that is best known for its role in constitutive heterochromatin formation and the repression of repetitive DNA elements. More recently, it has become evident that H3K9me3 is also deposited at certain loci in a tissue-specific manner and plays important roles in regulating cell identity. Notably, H3K9me3 can repress genes encoding silencing factors, pointing to a fundamental principle of repressive chromatin auto-regulation. Interestingly, recent studies have shown that H3K9me3 deposition requires protein SUMOylation in different contexts, suggesting that the SUMO pathway functions as an important module in gene silencing and heterochromatin formation. In this Review, we discuss the role of H3K9me3 in gene regulation in various systems and the molecular mechanisms that guide the silencing machinery to target loci.

Argonaute (Ago) proteins are the key players in RNA interference in eukaryotes, where they function as RNA-guided RNA endonucleases. Prokaryotic Argonautes (pAgos) are much more diverse than their eukaryotic counterparts but their cellular functions and mechanisms of action remain largely unknown. Some pAgos were shown to use small DNA guides for endonucleolytic cleave of complementary DNA in vitro. However, previously studied pAgos from thermophilic prokaryotes function at elevated temperatures which limits their potential use as a tool in genomic applications. Here, we describe two pAgos from mesophilic bacteria, Clostridium butyricum (CbAgo) and Limnothrix rosea (LrAgo), that act as DNA-guided DNA nucleases at physiological temperatures. In contrast to previously studied pAgos, CbAgo and LrAgo can use not only 5’-phosphorylated but also 5’-hydroxyl DNA guides, with diminished precision of target cleavage. Both LrAgo and CbAgo can tolerate guide/target mismatches in the seed region, but are sensitive to mismatches in the 3’-guide region. CbAgo is highly active under a wide range of conditions and can be used for programmable endonucleolytic cleavage of both single-stranded and double-stranded DNA substrates at moderate temperatures. The biochemical characterization of mesophilic pAgo proteins paths the way for their use for DNA manipulations both in vitro and in vivo.

The piRNA pathway is an adaptive mechanism that maintains genome stability by repression of selfish genomic elements. In the male germline of Drosophila melanogaster repression of Stellate genes by piRNAs generated from Supressor of Stellate (Su(Ste)) locus is required for male fertility, but both Su(Ste) piRNAs and their targets are absent in other Drosophila species. We found that D. melanogaster genome contains multiple X-linked non-coding genomic repeats that have sequence similarity to the protein-coding host gene vasa. In the male germline, these vasa-related AT-chX repeats produce abundant piRNAs that are antisense to vasa; however, vasa mRNA escapes silencing due to imperfect complementarity to AT-chX piRNAs. Unexpectedly, we discovered AT-chX piRNAs target vasa of Drosophila mauritiana in the testes of interspecies hybrids. In the majority of hybrid flies, the testes were strongly reduced in size and germline content. A minority of hybrids maintained wild-type array of premeiotic germ cells in the testes, but in them harmful Stellate genes were derepressed due to the absence of Su(Ste) piRNAs, and meiotic failures were observed. Thus, the piRNA pathway contributes to reproductive isolation between D. melanogaster and closely related species, causing hybrid male sterility via misregulation of two different host protein factors.

Regulation of transcription is the main mechanism responsible for precise control of gene expression. Whereas the majority of transcriptional regulation is mediated by DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, Piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. Here, we show that in Drosophila the small ubiquitin-like protein SUMO and the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase effector. We propose that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites.

Regulation of transcription is the main mechanism responsible for precise control of gene expression. While the majority of transcriptional regulation is mediated by a multitude of DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. Here we show that in Drosophila the small ubiquitin-like protein SUMO and the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase effector. We propose that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites.Highlights-piRNA-induced transposon silencing requires SUMO and the SUMO E3 ligase Su(var)2-10-Su(var)2-10 links the target recognition complex to the silencing effector-Su(var)2-10 binds the piRNA-guided target recognition complex and deposits SUMO on target chromatin-Su(var)2-10 induces SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase to target loci

Chromatin is critical for genome compaction and gene expression. On a coarse scale, the genome is divided into euchromatin, which harbors the majority of genes and is enriched in active chromatin marks, and heterochromatin, which is gene-poor but repeat-rich. The conserved molecular hallmark of heterochromatin is the H3K9me3 modification, which is associated with gene silencing. We found that in Drosophila deposition of most of the H3K9me3 mark depends on SUMO and the SUMO-ligase Su(var)2–10, which recruits the histone methyltransferase complex SetDB1/Wde. In addition to repressing repeats, H3K9me3 also influences expression of both hetero- and euchromatic host genes. High H3K9me3 levels in heterochromatin are required to suppress spurious transcription and ensure proper gene expression. In euchromatin, a set of conserved genes is repressed by Su(var)2–10/SetDB1-induced H3K9 trimethylation ensuring tissue-specific gene expression. Several components of heterochromatin are themselves repressed by this pathway providing a negative feedback mechanism to ensure chromatin homeostasis.

The eukaryotic Ago proteins and the RNA interference pathways they are involved in are widely used as a powerful tool in research and as potential therapeutics. In contrast, the properties and functions of prokaryotic Ago (pAgo) proteins have remained poorly understood. Understanding the diversity and functions of pAgos holds a huge potential for discovery of new cellular pathways and novel tools for genome manipulations. Only few pAgos have been characterized by structural or biochemical approaches, while previous genomic studies discovered about 300 proteins in archaeal and eubacterial genomes. Since that time the number of bacterial strains with sequenced genomes has greatly expanded, and many previously sequenced genomes have been revised. We undertook comprehensive analysis of pAgo proteins in sequenced genomes and almost tripled the number of known genes of this family. Our research thus forms a foundation for further experimental characterization of pAgo functions that will be important for understanding of the basic biology of these proteins and their adoption as a potential tool for genome engineering in the future., Members of the ancient family of Argonaute (Ago) proteins are present in all domains of life. The common feature of Ago proteins is the ability to bind small nucleic acid guides and use them for sequence-specific recognition—and sometimes cleavage—of complementary targets. While eukaryotic Ago (eAgo) proteins are key players in RNA interference and related pathways, the properties and functions of these proteins in archaeal and bacterial species have just started to emerge. We undertook comprehensive exploration of prokaryotic Ago (pAgo) proteins in sequenced genomes and revealed their striking diversity in comparison with eAgos. Many pAgos contain divergent variants of the conserved domains involved in interactions with nucleic acids, while having extra domains that are absent in eAgos, suggesting that they might have unusual specificities in the nucleic acid recognition and cleavage. Many pAgos are associated with putative nucleases, helicases, and DNA binding proteins in the same gene or operon, suggesting that they are involved in target processing. The great variability of pAgos revealed by our analysis opens new ways for exploration of their functions in host cells and for their use as potential tools in genome editing.

Noncoding RNAs play essential roles in genetic regulation in all organisms. In eukaryotic cells, many small non-coding RNAs act in complex with Argonaute proteins and regulate gene expression by recognizing complementary RNA targets. The complexes of Argonaute proteins with small RNAs also play a key role in silencing of mobile genetic elements and, in some cases, viruses. These processes are collectively called RNA interference. RNA interference is a powerful tool for specific gene silencing in both basic research and therapeutic applications. Argonaute proteins are also found in prokaryotic organisms. Recent studies have shown that prokaryotic Argonautes can also cleave their target nucleic acids, in particular DNA. This activity of prokaryotic Argonautes might potentially be used to edit eukaryotic genomes. However, the molecular mechanisms of small nucleic acid biogenesis and the functions of Argonaute proteins, in particular in bacteria and archaea, remain largely unknown. Here we briefly review available data on the RNA interference processes and Argonaute proteins in eukaryotes and prokaryotes.

Endothelial cell (EC)-enriched protein coding genes, such as endothelial nitric oxide synthase (eNOS), define quintessential EC-specific physiologic functions. It is not clear whether long noncoding RNAs (lncRNAs) also define cardiovascular cell type-specific phenotypes, especially in the vascular endothelium. Here, we report the existence of a set of EC-enriched lncRNAs and define a role for spliced-transcript endothelial-enriched lncRNA (STEEL) in angiogenic potential, macrovascular/microvascular identity, and shear stress responsiveness. STEEL is expressed from the terminus of the HOXD locus and is transcribed antisense to HOXD transcription factors. STEEL RNA increases the number and integrity of de novo perfused microvessels in an in vivo model and augments angiogenesis in vitro. The STEEL RNA is polyadenylated, nuclear enriched, and has microvascular predominance. Functionally, STEEL regulates a number of genes in diverse ECs. Of interest, STEEL up-regulates both eNOS and the transcription factor Kruppel-like factor 2 (KLF2), and is subject to feedback inhibition by both eNOS and shear-augmented KLF2. Mechanistically, STEEL up-regulation of eNOS and KLF2 is transcriptionally mediated, in part, via interaction of chromatin-associated STEEL with the poly-ADP ribosylase, PARP1. For instance, STEEL recruits PARP1 to the KLF2 promoter. This work identifies a role for EC-enriched lncRNAs in the phenotypic adaptation of ECs to both body position and hemodynamic forces and establishes a newer role for lncRNAs in the transcriptional regulation of EC identity.

Visually-guided behaviour is constrained by the capacity of the visual system to resolve detail. This is, in turn, limited by the spatial resolution and contrast sensitivity of the underlying visual system. Because these properties are interdependent and vary non-uniformly, it is only possible to fully understand the limits of a specific visually guided behaviour when they are investigated in combination. To understand the visual limits of flight control in bees, which rely heavily on vision to control flight, and to explore whether they vary between species, we tested how changes in spatial resolution and contrast sensitivity affect the speed and position control of the Asian and European honeybees (Apis cerana and A. mellifera). Despite their apparent similarity, we found some interesting and surprising differences between the visual limits of these species. While the effect of spatial frequency and contrast on position control is similar between the species, ground speed is differently affected by these variables. A comparison with published data from the bumblebee Bombus terrestris reveals further differences. The visual resolution that limits the detection and use of optic flow for flight control in both species of honeybees is lower than previously anatomically determined resolution and differs from object detection limits of A. mellifera, providing evidence that the limits of spatial resolution and contrast sensitivity are highly tuned to the particular behavioural task of a species.

The PIWI-interacting RNA (piRNA) pathway is a conserved defense system that protects the genome integrity of the animal germline from deleterious transposable elements. Targets of silencing are recognized by small noncoding piRNAs that are processed from long precursor molecules. Although piRNAs and other classes of small noncoding RNAs, such as miRNAs and small interfering (si)RNAs, interact with members of the same family of Argonaute (Ago) proteins and their function in target repression is similar, the biogenesis of piRNAs differs from those of the other two small RNAs. Recently, many aspects of piRNA biogenesis have been revealed in Drosophila melanogaster. In this review, we elaborate on piRNA biogenesis in Drosophila somatic and germline cells. We focus on the mechanisms by which piRNA precursor transcription is regulated and highlight recent work that has advanced our understanding of piRNA precursor processing to mature piRNAs. We finish by discussing current models to the still unresolved question of how piRNA precursors are selected and channeled into the processing machinery.

Summary In developing male germ cells, prospermatogonia, two Piwi proteins, MILI and MIWI2, use Piwi-interacting RNA (piRNA) guides to repress transposable element (TE) expression and ensure genome stability and proper gametogenesis. In addition to their roles in post-transcriptional TE repression, both proteins are required for DNA methylation of TE sequences. Here, we analyzed the effect of Miwi2 deficiency on piRNA biogenesis and transposon repression. Miwi2 deficiency had only a minor impact on piRNA biogenesis; however, the piRNA profile of Miwi2-knockout mice indicated overexpression of several LINE1 TE families that led to activation of the ping-pong piRNA cycle. Furthermore, we found that MILI and MIWI2 have distinct functions in TE repression in the nucleus. MILI is responsible for DNA methylation of a larger subset of TE families than MIWI2 is, suggesting that the proteins have independent roles in establishing DNA methylation patterns., Graphical abstract

Summary: Prokaryotic Argonaute (Ago) proteins were recently shown to target foreign genetic elements, thus making them a perfect model for studies of interference mechanisms. Here, we study interactions of Rhodobacter sphaeroides Ago (RsAgo) with guide RNA (gRNA) and fully complementary or imperfect target DNA (tDNA) using biochemical and structural approaches. We show that RsAgo can specifically recognize both the first nucleotide in gRNA and complementary nucleotide in tDNA, and both interactions contribute to nucleic acid binding. Non-canonical pairs and bulges on the target strand can be accommodated by RsAgo with minimal perturbation of the duplex but significantly reduce RsAgo affinity to tDNA. Surprisingly, mismatches between gRNA and tDNA induce dissociation of the guide-target duplex from RsAgo. Our results reveal plasticity in the ability of Ago proteins to accommodate helical imperfections, show how this might affect the efficiency of RNA silencing, and suggest a potential mechanism for guide release and Ago recycling. : Prokaryotic Argonaute proteins (pAgos) target foreign genetic elements. Liu et al. structurally and functionally characterize interactions of pAgo with guide RNA and fully complementary or imperfectly targeted DNA, reveal plasticity in the ability of Ago proteins to accommodate helical imperfections, and suggest a potential mechanism for guide release. Keywords: Rhodobacter sphaeroides Argonaute, RsAgo, guide RNA, target DNA, RNA-DNA heteroduplex, non-canonical base pairs and bulges

Many insects rely on vision to find food, to return to their nest and to carefully control their flight between these two locations. The amount of information available to support these tasks is, in part, dictated by the spatial resolution and contrast sensitivity of their visual systems. Here, we investigate the absolute limits of these visual properties for visually guided position and speed control in Bombus terrestris. Our results indicate that the limit of spatial vision in the translational motion detection system of B. terrestris lies at 0.21 cycles deg−1 with a peak contrast sensitivity of at least 33. In the perspective of earlier findings, these results indicate that bumblebees have higher contrast sensitivity in the motion detection system underlying position control than in their object discrimination system. This suggests that bumblebees, and most likely also other insects, have different visual thresholds depending on the behavioral context. Electronic supplementary material The online version of this article (doi:10.1007/s00359-017-1212-6) contains supplementary material, which is available to authorized users.

Transposable elements (TEs) occupy a large fraction of metazoan genomes and pose a constant threat to genomic integrity. This threat is particularly critical in germ cells, as changes in the genome that are induced by TEs will be transmitted to the next generation. Small noncoding piwi-interacting RNAs (piRNAs) recognize and silence a diverse set of TEs in germ cells. In mice, piRNA-guided transposon repression correlates with establishment of CpG DNA methylation on their sequences, yet the mechanism and the spectrum of genomic targets of piRNA silencing are unknown. Here we show that in addition to DNA methylation, the piRNA pathway is required to maintain a high level of the repressive H3K9me3 histone modification on long interspersed nuclear elements (LINEs) in germ cells. piRNA-dependent chromatin repression targets exclusively full-length elements of actively transposing LINE families, demonstrating the remarkable ability of the piRNA pathway to recognize active elements among the large number of genomic transposon fragments.

Eukaryotic Argonautes bind small RNAs and use them as guides to find complementary RNA targets and induce gene silencing. Though homologs of eukaryotic Argonautes are present in many bacteria and archaea, their small RNA partners and functions are unknown. We found that the Argonaute of Rhodobacter sphaeroides (RsAgo) associates with 15-19 nt RNAs that correspond to the majority of transcripts. RsAgo also binds single-stranded 22-24 nt DNA molecules that are complementary to the small RNAs and enriched in sequences derived from exogenous plasmids as well as genome-encoded foreign nucleic acids such as transposons and phage genes. Expression of RsAgo in the heterologous E. coli system leads to formation of plasmid-derived small RNA and DNA and plasmid degradation. In a R. sphaeroides mutant lacking RsAgo, expression of plasmid-encoded genes is elevated. Our results indicate that RNAi-related processes found in eukaryotes are also conserved in bacteria and target foreign nucleic acids.

Tandem Stellate genes organized into two clusters in heterochromatin and euchromatin of the X-chromosome are part of the Ste-Su(Ste) genetic system required for maintenance of male fertility and reproduction of Drosophila melanogaster. Stellate genes encode a regulatory subunit of protein kinase CK2 and are the main targets of germline-specific piRNA-silencing; their derepression leads to appearance of protein crystals in spermatocytes, meiotic disturbances, and male sterility. A short promoter region of 134 bp appears to be sufficient for testis-specific transcription of Stellate, and it contains three closely located cis-regulatory elements called E-boxes. By using reporter analysis, we confirmed a strong functionality of the E-boxes in the Stellate promoter for in vivo transcription. Using selective mutagenesis, we have shown that the presence of the central E-box 2 is preferable to maintain a high-level testis-specific transcription of the reporter gene under the Stellate promoter. The Stellate promoter provides transcription even in heterochromatin, and corresponding mRNAs are translated with the generation of full-size protein products in case of disturbances in the piRNA-silencing process. We have also shown for the first time that the activity of the Stellate promoter is determined by chromatin context of the X-chromosome in male germinal cells, and it increases at about twofold when relocating in autosomes.

Small non-coding RNAs called piRNAs serve as guides for an adaptable immune system that represses transposable elements in germ cells of Metazoa. In Drosophila the RDC complex, composed ofRhino, Deadlock and Cutoff (Cuff) bind chromatin of dual-strand piRNA clusters, special genomic regions, which encode piRNA precursors. The RDC complex is required for transcription of piRNA precursors, though the mechanism by which it licenses transcription remained unknown. Here, we show that Cuff prevents premature termination of RNA polymerase II. Cuff prevents cleavage of nascent RNA at poly(A) sites by interfering with recruitment of thecleavage and polyadenylation specificity factor (CPSF) complex. Cuff also protects processed transcripts from degradation by the exonuclease Rat1. Our work reveals a conceptually different mechanism of transcriptional enhancement. In contrast to other factors that regulate termination by binding to specific signals on nascent RNA, the RDC complex inhibits termination in a chromatin-dependent and sequence-independent manner.

The conserved THO/TREX (transcription/export) complex is critical for pre-mRNA processing and mRNA nuclear export. In metazoa, TREX is loaded on nascent RNA transcribed by RNA polymerase II in a splicing-dependent fashion; however, how TREX functions is poorly understood. Here we show that Thoc5 and other TREX components are essential for the biogenesis of piRNA, a distinct class of small noncoding RNAs that control expression of transposable elements (TEs) in the Drosophila germline. Mutations in TREX lead to defects in piRNA biogenesis, resulting in derepression of multiple TE families, gametogenesis defects, and sterility. TREX components are enriched on piRNA precursors transcribed from dual-strand piRNA clusters and colocalize in distinct nuclear foci that overlap with sites of piRNA transcription. The localization of TREX in nuclear foci and its loading on piRNA precursor transcripts depend on Cutoff, a protein associated with chromatin of piRNA clusters. Finally, we show that TREX is required for accumulation of nascent piRNA precursors. Our study reveals a novel splicing-independent mechanism for TREX loading on nascent RNA and its importance in piRNA biogenesis.

Eukaryotes use several classes of small RNA molecules to guide diverse protein machineries to target messenger RNA. The role of small RNA in post-transcriptional regulation of mRNA stability and translation is now well established. Small RNAs can also guide sequence-specific modification of chromatin structure and thus contribute to establishment and maintenance of distinct chromatin domains. In this review we summarize the model for the inter-dependent interaction between small RNA and chromatin that has emerged from studies on fission yeast and plants. We focus on recent results that link a distinct class of small RNAs, the piRNAs, to chromatin regulation in animals.

Bombus terrestris is one of the most commonly used insect models to investigate visually guided behavior and spatial vision in particular. Two fundamental measures of spatial vision are spatial resolution and contrast sensitivity. In this study, we report the threshold of spatial resolution in B. terrestris and characterize the contrast sensitivity function of the bumblebee visual system for a dual choice discrimination task. We trained bumblebees in a Y-maze experimental set-up to associate a vertical sinusoidal grating with a sucrose reward, and a horizontal grating with absence of a reward. Using a logistic psychometric function, we estimated a resolution threshold of 0.21 cycles deg(-1) of visual angle. This resolution is in the same range but slightly lower than that found in honeybees (Apis mellifera and A. cerana) and another bumblebee species (B. impatiens). We also found that the contrast sensitivity of B. terrestris was 1.57 for the spatial frequency 0.090 cycles deg(-1) and 1.26 for 0.18 cycles deg(-1).

Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.

Argonaute proteins are central players in small RNA-mediated silencing mechanisms such as RNA interference (RNAi), microRNA repression and piRNA-mediated transposon silencing. In eukaryotes, Argonautes bind small RNAs that guide them to RNA targets in order to regulate gene expression and repress invasive genomic elements. Although Argonaute proteins are conserved in all life forms from bacteria to eukaryotes, until now studies have focused on the biological functions of eukaryotic Argonautes. Here we highlight two recent studies that discover the functions of prokaryotic Argonautes in defence against exogenous DNA.

Germline genomic methylation is essential for gamete identity and integrity in mammals. The study by Kuramochi-Miyagawa and colleagues (908–917) in the previous issue of Genes & Development links the process of DNA methylation-dependent repression of retrotranspons with the presence of piwi-interacting RNAs (piRNAs) in fetal male germ cells undergoing de novo methylation.

Over evolutionary time, many genes undergo duplication and one copy accumulates mutations that render it non-functional. These 'pseudogenes' are generally thought to be rather uninteresting, dead-end pieces of the genome. Yet there now appears to be more to it than that. Two groups report in this issue on pseudogenes that can in fact influence gene expression. The mechanism involves pairing of RNA antisense transcripts from pseudogenes with the mRNAs of protein-coding genes, forming a duplex RNA that is processed into endogenous siRNAs. Over evolutionary time genes can undergo duplication, and may accumulate mutations that render them non-functional pseudogenes, which are thought to be uninteresting. This study (and that of the group of Sasaki) shows that pseudogenes can in fact influence gene expression. Pseudogenes populate the mammalian genome as remnants of artefactual incorporation of coding messenger RNAs into transposon pathways1. Here we show that a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes. These endo-siRNAs are often processed from double-stranded RNAs formed by hybridization of spliced transcripts from protein-coding genes to antisense transcripts from homologous pseudogenes. An inverted repeat pseudogene can also generate abundant small RNAs directly. A second class of endo-siRNAs may enforce repression of mobile genetic elements, acting together with Piwi-interacting RNAs. Loss of Dicer, a protein integral to small RNA production, increases expression of endo-siRNA targets, demonstrating their regulatory activity. Our findings indicate a function for pseudogenes in regulating gene expression by means of the RNA interference pathway and may, in part, explain the evolutionary pressure to conserve argonaute-mediated catalysis in mammals.

During the past several years, it has become clear that small RNAs guard germ cell genomes from the activity of mobile genetic elements. Indeed, in mammals, a class of small RNAs, known as Piwi-interacting RNAs (piRNAs), forms an innate immune system that discriminates transposons from endogenous genes and selectively silences the former. piRNAs enforce silencing by directing transposon DNA methylation during male germ cell development. As such, piRNAs represent perhaps the only currently known sequence-specific factor for deposition of methylcytosine in mammals. The three mammalian Piwi proteins Miwi2, Mili, and Miwi are required at different stages of germ cell development. Moreover, distinct classes of piRNAs are expressed in developmental waves, with particular generative loci and different sequence content distinguishing piRNAs populations in embryonic germ cells from those that appear during meiosis. Although our understanding of Piwi proteins and piRNA biology have deepened substantially during the last several years, major gaps still exist in our understanding of these enigmatic RNA species.

Nearly half of the mammalian genome is composed of repeated sequences. In Drosophila , Piwi proteins exert control over transposons. However, mammalian Piwi proteins, MIWI and MILI, partner with Piwi-interacting RNAs (piRNAs) that are depleted of repeat sequences, which raises questions about a role for mammalian Piwi's in transposon control. A search for murine small RNAs that might program Piwi proteins for transposon suppression revealed developmentally regulated piRNA loci, some of which resemble transposon master control loci of Drosophila . We also find evidence of an adaptive amplification loop in which MILI catalyzes the formation of piRNA 5′ ends. Mili mutants derepress LINE-1 (L1) and intracisternal A particle and lose DNA methylation of L1 elements, demonstrating an evolutionarily conserved role for PIWI proteins in transposon suppression.