The chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus uncovers patterns of genome evolution in mosquitoes.

BACKGROUND: Understanding genome organization and evolution is important for species involved in transmission of human diseases, such as mosquitoes. Anophelinae and Culicinae subfamilies of mosquitoes show striking differences in genome sizes, sex chromosome arrangements, behavior, and ability to transmit pathogens. However, the genomic basis of these differences is not fully understood. METHODS: In this study, we used a combination of advanced genome technologies such as Oxford Nanopore Technology sequencing, Hi-C scaffolding, Bionano, and cytogenetic mapping to develop an improved chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus. RESULTS: We then used this assembly to annotate odorant receptors, odorant binding proteins, and transposable elements. A genomic region containing male-specific sequences on chromosome 1 and a polymorphic inversion on chromosome 3 were identified in the Cx. quinquefasciatus genome. In addition, the genome of Cx. quinquefasciatus was compared with the genomes of other mosquitoes such as malaria vectors An. coluzzi and An. albimanus, and the vector of arboviruses Ae. aegypti. Our work confirms significant expansion of the two chemosensory gene families in Cx. quinquefasciatus, as well as a significant increase and relocation of the transposable elements in both Cx. quinquefasciatus and Ae. aegypti relative to the Anophelines. Phylogenetic analysis clarifies the divergence time between the mosquito species. Our study provides new insights into chromosomal evolution in mosquitoes and finds that the X chromosome of Anophelinae and the sex-determining chromosome 1 of Culicinae have a significantly higher rate of evolution than autosomes. CONCLUSION: The improved Cx. quinquefasciatus genome assembly uncovered new details of mosquito genome evolution and has the potential to speed up the development of novel vector control strategies.

Fraternity of old-timers: How ubiquitin regulates miRNA functions.

miRNA-mediated gene repression and ubiquitin-dependent processes are among the oldest and most versatile mechanisms that control multiple molecular pathways, rather than just protein turnover. These systems were discovered decades ago and have become among the most studied. All systems within cells are interconnected, and these two are no exception: the plethora of studies have demonstrated that the activity of the miRNAs system depends on players of the ubiquitin-centered universe of processes, and vice versa. This review focuses on recent progress that highlights that very similar mechanisms of regulation of miRNAs by ubiquitin-related processes are likely to be found in distantly related species, including animals, plants, and viruses. Most of them occur through the ubiquitination of Argonaute proteins, but some of the other miRNA system factors are also regulated. This suggests that their regulatory relationships are either ancient evolutionary acquisitions or have arisen independently in different kingdoms.

Bacterial Argonaute nucleases reveal different modes of DNA targeting in vitro and in vivo.

Prokaryotic Argonaute proteins (pAgos) are homologs of eukaryotic Argonautes (eAgos) and are also thought to play a role in cell defense against invaders. However, pAgos are much more diverse than eAgos and little is known about their functional activities and target specificities in vivo. Here, we describe five pAgos from mesophilic bacteria that act as programmable DNA endonucleases and analyze their ability to target chromosomal and invader DNA. In vitro, the analyzed proteins use small guide DNAs for precise cleavage of single-stranded DNA at a wide range of temperatures. Upon their expression in Escherichia coli, all five pAgos are loaded with small DNAs preferentially produced from plasmids and chromosomal regions of replication termination. One of the tested pAgos, EmaAgo from Exiguobacterium marinum, can induce DNA interference between homologous sequences resulting in targeted processing of multicopy plasmid and genomic elements. EmaAgo also protects bacteria from bacteriophage infection, by loading phage-derived guide DNAs and decreasing phage DNA content and phage titers. Thus, the ability of pAgos to target multicopy elements may be crucial for their protective function. The wide spectrum of pAgo activities suggests that they may have diverse functions in vivo and paves the way for their use in biotechnology.

Programmable RNA targeting by bacterial Argonaute nucleases with unconventional guide binding and cleavage specificity.

Argonaute proteins are programmable nucleases that have defense and regulatory functions in both eukaryotes and prokaryotes. All known prokaryotic Argonautes (pAgos) characterized so far act on DNA targets. Here, we describe a new class of pAgos that uniquely use DNA guides to process RNA targets. The biochemical and structural analysis of Pseudooceanicola lipolyticus pAgo (PliAgo) reveals an unusual organization of the guide binding pocket that does not rely on divalent cations and the canonical set of contacts for 5'-end interactions. Unconventional interactions of PliAgo with the 5'-phosphate of guide DNA define its new position within pAgo and shift the site of target RNA cleavage in comparison with known Argonautes. The specificity for RNA over DNA is defined by ribonucleotide residues at the cleavage site. The analysed pAgos sense mismatches and modifications in the RNA target. The results broaden our understanding of prokaryotic defense systems and extend the spectrum of programmable nucleases with potential use in RNA technology.

Extended disordered regions of ribosome-associated NAC proteins paralogs belong only to the germline in Drosophila melanogaster.

The nascent polypeptide-associated complex (NAC) consisting of α- and ß-subunits is an essential ribosome-associated protein conserved in eukaryotes. NAC is a ubiquitously expressed co-translational regulator of nascent protein folding and sorting providing for homeostasis of cellular proteins. Here we report on discovering the germline-specific NACαß paralogs (gNACs), whose ß-subunits, non-distinguishable by ordinary immunodetection, are encoded by five highly homologous gene copies, while the α-subunit is encoded by a single αNAC gene. The gNAC expression is detected in the primordial embryonic and adult gonads via immunostaining. The germline-specific α and ß subunits differ from the ubiquitously expressed paralogs by the extended intrinsically disordered regions (IDRs) acquired at the N- and C-termini of the coding regions, predicted to be phosphorylated. The presence of distinct phosphorylated isoforms of gNAC-ß subunits is confirmed by comparing of their profiles by 2D-isoeletrofocusing resolution before and after phosphatase treatment of testis ribosomes. We revealed that the predicted S/T sites of phosphorylation in the individual orthologous IDRs of gNAC-ß sequences of Drosophila species are positionally conserved despite these disordered regions are drastically different. We propose the IDR-dependent molecular crowding and specific coordination of NAC and other proteostasis regulatory factors at the ribosomes of germinal cells. Our findings imply that there may be a functional crosstalk between the germinal and ubiquitous α- and ß-subunits based on assessing their depletion effects on the fly viability and gonad development.

Noncanonical prokaryotic X family DNA polymerases lack polymerase activity and act as exonucleases.

The X family polymerases (PolXs) are specialized DNA polymerases that are found in all domains of life. While the main representatives of eukaryotic PolXs, which have dedicated functions in DNA repair, were studied in much detail, the functions and diversity of prokaryotic PolXs have remained largely unexplored. Here, by combining a comprehensive bioinformatic analysis of prokaryotic PolXs and biochemical experiments involving selected recombinant enzymes, we reveal a previously unrecognized group of PolXs that seem to be lacking DNA polymerase activity. The noncanonical PolXs contain substitutions of the key catalytic residues and deletions in their polymerase and dNTP binding sites in the palm and fingers domains, but contain functional nuclease domains, similar to canonical PolXs. We demonstrate that representative noncanonical PolXs from the Deinococcus genus are indeed inactive as DNA polymerases but are highly efficient as 3'-5' exonucleases. We show that both canonical and noncanonical PolXs are often encoded together with the components of the non-homologous end joining pathway and may therefore participate in double-strand break repair, suggesting an evolutionary conservation of this PolX function. This is a remarkable example of polymerases that have lost their main polymerase activity, but retain accessory functions in DNA processing and repair.

DNA targeting and interference by a bacterial Argonaute nuclease.

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.

Nuclear Ccr4-Not mediates the degradation of telomeric and transposon transcripts at chromatin in the Drosophila germline.

Ccr4-Not is a highly conserved complex involved in cotranscriptional RNA surveillance pathways in yeast. In Drosophila, Ccr4-Not is linked to the translational repression of miRNA targets and the posttranscriptional control of maternal mRNAs during oogenesis and embryonic development. Here, we describe a new role for the Ccr4-Not complex in nuclear RNA metabolism in the Drosophila germline. Ccr4 depletion results in the accumulation of transposable and telomeric repeat transcripts in the fraction of chromatin-associated RNA; however, it does not affect small RNA levels or the heterochromatin state of the target loci. Nuclear targets of Ccr4 mainly comprise active full-length transposable elements (TEs) and telomeric and subtelomeric repeats. Moreover, Ccr4-Not foci localize at telomeres in a Piwi-dependent manner, suggesting a functional relationship between these pathways. Indeed, we detected interactions between the components of the Ccr4-Not complex and piRNA machinery, which indicates that these pathways cooperate in the nucleus to recognize and degrade TE transcripts at transcription sites. These data reveal a new layer of transposon control in the germline, which is critical for the maintenance of genome integrity.

Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea.

Argonaute (Ago) proteins are 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 cleavage 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 comparison with previously studied pAgos, CbAgo and LrAgo do not show strong preferences for the 5'-nucleotide in guide DNA and can use not only 5'-phosphorylated but also 5'-hydroxyl DNA guides. Both CbAgo and LrAgo can tolerate guide/target mismatches in the seed region, but are sensitive to mismatches in the 3'-guide region. Both pAgos can perform programmable endonucleolytic cleavage of double-stranded DNA substrates, showing enhanced activity at AT-rich regions and at elevated temperatures. The biochemical characterization of mesophilic pAgo proteins paves the way for their use for DNA manipulations both in vitro and in vivo.

The Integrity of piRNA Clusters is Abolished by Insulators in the Drosophila Germline.

Piwi-interacting RNAs (piRNAs) control transposable element (TE) activity in the germline. piRNAs are produced from single-stranded precursors transcribed from distinct genomic loci, enriched by TE fragments and termed piRNA clusters. The specific chromatin organization and transcriptional regulation of Drosophila germline-specific piRNA clusters ensure transcription and processing of piRNA precursors. TEs harbour various regulatory elements that could affect piRNA cluster integrity. One of such elements is the suppressor-of-hairy-wing (Su(Hw))-mediated insulator, which is harboured in the retrotransposon gypsy. To understand how insulators contribute to piRNA cluster activity, we studied the effects of transgenes containing gypsy insulators on local organization of endogenous piRNA clusters. We show that transgene insertions interfere with piRNA precursor transcription, small RNA production and the formation of piRNA cluster-specific chromatin, a hallmark of which is Rhino, the germline homolog of the heterochromatin protein 1 (HP1). The mutations of Su(Hw) restored the integrity of piRNA clusters in transgenic strains. Surprisingly, Su(Hw) depletion enhanced the production of piRNAs by the domesticated telomeric retrotransposon TART, indicating that Su(Hw)-dependent elements protect TART transcripts from piRNA processing machinery in telomeres. A genome-wide analysis revealed that Su(Hw)-binding sites are depleted in endogenous germline piRNA clusters, suggesting that their functional integrity is under strict evolutionary constraints.

The Expanded Universe of Prokaryotic Argonaute Proteins.

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.IMPORTANCE 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.

Key role of piRNAs in telomeric chromatin maintenance and telomere nuclear positioning in Drosophila germline.

BACKGROUND: Telomeric small RNAs related to PIWI-interacting RNAs (piRNAs) have been described in various eukaryotes; however, their role in germline-specific telomere function remains poorly understood. Using a Drosophila model, we performed an in-depth study of the biogenesis of telomeric piRNAs and their function in telomere homeostasis in the germline. RESULTS: To fully characterize telomeric piRNA clusters, we integrated the data obtained from analysis of endogenous telomeric repeats, as well as transgenes inserted into different telomeric and subtelomeric regions. The small RNA-seq data from strains carrying telomeric transgenes demonstrated that all transgenes belong to a class of dual-strand piRNA clusters; however, their capacity to produce piRNAs varies significantly. Rhino, a paralog of heterochromatic protein 1 (HP1) expressed exclusively in the germline, is associated with all telomeric transgenes, but its enrichment correlates with the abundance of transgenic piRNAs. It is likely that this heterogeneity is determined by the sequence peculiarities of telomeric retrotransposons. In contrast to the heterochromatic non-telomeric germline piRNA clusters, piRNA loss leads to a dramatic decrease in HP1, Rhino, and trimethylated histone H3 lysine 9 in telomeric regions. Therefore, the presence of piRNAs is required for the maintenance of telomere chromatin in the germline. Moreover, piRNA loss causes telomere translocation from the nuclear periphery toward the nuclear interior but does not affect telomere end capping. Analysis of the telomere-associated sequences (TASs) chromatin revealed strong tissue specificity. In the germline, TASs are enriched with HP1 and Rhino, in contrast to somatic tissues, where they are repressed by Polycomb group proteins. CONCLUSIONS: piRNAs play an essential role in the assembly of telomeric chromatin, as well as in nuclear telomere positioning in the germline. Telomeric arrays and TASs belong to a unique type of Rhino-dependent piRNA clusters with transcripts that serve simultaneously as piRNA precursors and as their only targets. Telomeric chromatin is highly sensitive to piRNA loss, implying the existence of a novel developmental checkpoint that depends on telomere integrity in the germline.

Transcriptional and chromatin changes accompanying de novo formation of transgenic piRNA clusters.

Expression of transposable elements in the germline is controlled by Piwi-interacting (pi) RNAs produced by genomic loci termed piRNA clusters and associated with Rhino, a heterochromatin protein 1 (HP1) homolog. Previously, we have shown that transgenes containing a fragment of the I retrotransposon form de novo piRNA clusters in the Drosophila germline providing suppression of I-element activity. We noted that identical transgenes located in different genomic sites vary considerably in piRNA production and classified them as "strong" and "weak" piRNA clusters. Here, we investigated what chromatin and transcriptional changes occur at the transgene insertion sites after their conversion into piRNA clusters. We found that the formation of a transgenic piRNA cluster is accompanied by activation of transcription from both genomic strands that likely initiates at multiple random sites. The chromatin of all transgene-associated piRNA clusters contain high levels of trimethylated lysine 9 of histone H3 (H3K9me3) and HP1a, whereas Rhino binding is considerably higher at the strong clusters. None of these chromatin marks was revealed at the "empty" sites before transgene insertion. Finally, we have shown that in the nucleus of polyploid nurse cells, the formation of a piRNA cluster at a given transgenic genomic copy works according to an "all-or-nothing" model: either there is high Rhino enrichment or there is no association with Rhino at all. As a result, genomic copies of a weak piRNA transgenic cluster show a mosaic association with Rhino foci, while the majority of strong transgene copies associate with Rhino and are hence involved in piRNA production.

Drosophila Model for the Analysis of Genesis of LIM-kinase 1-Dependent Williams-Beuren Syndrome Cognitive Phenotypes: INDELs, Transposable Elements of the Tc1/Mariner Superfamily and MicroRNAs.

Genomic disorders, the syndromes with multiple manifestations, may occur sporadically due to unequal recombination in chromosomal regions with specific architecture. Therefore, each patient may carry an individual structural variant of DNA sequence (SV) with small insertions and deletions (INDELs) sometimes less than 10 bp. The transposable elements of the Tc1/mariner superfamily are often associated with hotspots for homologous recombination involved in human genetic disorders, such as Williams Beuren Syndromes (WBS) with LIM-kinase 1-dependent cognitive defects. The Drosophila melanogaster mutant agnts3 has unusual architecture of the agnostic locus harboring LIMK1: it is a hotspot of chromosome breaks, ectopic contacts, underreplication, and recombination. Here, we present the analysis of LIMK1-containing locus sequencing data in agnts3 and three D. melanogaster wild-type strains-Canton-S, Berlin, and Oregon-R. We found multiple strain-specific SVs, namely, single base changes and small INDEls. The specific feature of agnts3 is 28 bp A/T-rich insertion in intron 1 of LIMK1 and the insertion of mobile S-element from Tc1/mariner superfamily residing ~460 bp downstream LIMK1 3'UTR. Neither of SVs leads to amino acid substitutions in agnts3 LIMK1. However, they apparently affect the nucleosome distribution, non-canonical DNA structure formation and transcriptional factors binding. Interestingly, the overall expression of miRNAs including the biomarkers for human neurological diseases, is drastically reduced in agnts3 relative to the wild-type strains. Thus, LIMK1 DNA structure per se, as well as the pronounced changes in total miRNAs profile, probably lead to LIMK1 dysregulation and complex behavioral dysfunctions observed in agnts3 making this mutant a simple plausible Drosophila model for WBS.

Piwi interacts with chromatin at nuclear pores and promiscuously binds nuclear transcripts in Drosophila ovarian somatic cells.

Piwi in a complex with Piwi-interacting RNAs (piRNAs) triggers transcriptional silencing of transposable elements (TEs) in Drosophila ovaries, thus ensuring genome stability. To do this, Piwi must scan the nascent transcripts of genes and TEs for complementarity to piRNAs. The mechanism of this scanning is currently unknown. Here we report the DamID-seq mapping of multiple Piwi-interacting chromosomal domains in somatic cells of Drosophila ovaries. These domains significantly overlap with genomic regions tethered to Nuclear Pore Complexes (NPCs). Accordingly, Piwi was coimmunoprecipitated with the component of NPCs Elys and with the Xmas-2 subunit of RNA transcription and export complex, known to interact with NPCs. However, only a small Piwi fraction has transient access to DNA at nuclear pores. Importantly, although 36% of the protein-coding genes overlap with Piwi-interacting domains and RNA-immunoprecipitation results demonstrate promiscuous Piwi binding to numerous genic and TE nuclear transcripts, according to available data Piwi does not silence these genes, likely due to the absence of perfect base-pairing between piRNAs and their transcripts.

Natural variation of piRNA expression affects immunity to transposable elements.

In the Drosophila germline, transposable elements (TEs) are silenced by PIWI-interacting RNA (piRNA) that originate from distinct genomic regions termed piRNA clusters and are processed by PIWI-subfamily Argonaute proteins. Here, we explore the variation in the ability to restrain an alien TE in different Drosophila strains. The I-element is a retrotransposon involved in the phenomenon of I-R hybrid dysgenesis in Drosophila melanogaster. Genomes of R strains do not contain active I-elements, but harbour remnants of ancestral I-related elements. The permissivity to I-element activity of R females, called reactivity, varies considerably in natural R populations, indicating the existence of a strong natural polymorphism in defense systems targeting transposons. To reveal the nature of such polymorphisms, we compared ovarian small RNAs between R strains with low and high reactivity and show that reactivity negatively correlates with the ancestral I-element-specific piRNA content. Analysis of piRNA clusters containing remnants of I-elements shows increased expression of the piRNA precursors and enrichment by the Heterochromatin Protein 1 homolog, Rhino, in weak R strains, which is in accordance with stronger piRNA expression by these regions. To explore the nature of the differences in piRNA production, we focused on two R strains, weak and strong, and showed that the efficiency of maternal inheritance of piRNAs as well as the I-element copy number are very similar in both strains. At the same time, germline and somatic uni-strand piRNA clusters generate more piRNAs in strains with low reactivity, suggesting the relationship between the efficiency of primary piRNA production and variable response to TE invasions. The strength of adaptive genome defense is likely driven by naturally occurring polymorphisms in the rapidly evolving piRNA pathway proteins. We hypothesize that hyper-efficient piRNA production is contributing to elimination of a telomeric retrotransposon HeT-A, which we have observed in one particular transposon-resistant R strain.

Telomeric Retrotransposon HeT-A Contains a Bidirectional Promoter that Initiates Divergent Transcription of piRNA Precursors in Drosophila Germline.

PIWI-interacting RNAs (piRNAs) provide the silencing of transposable elements in the germline. Drosophila telomeres are maintained by transpositions of specialized telomeric retroelements. piRNAs generated from sense and antisense transcripts of telomeric elements provide telomere length control in the germline. Previously, we have found that antisense transcription of the major telomeric retroelement HeT-A is initiated upstream of the HeT-A sense transcription start site. Here, we performed a deletion analysis of the HeT-A promoter and show that common regulatory elements are shared by sense and antisense promoters of HeT-A. Therefore, the HeT-A promoter is a bidirectional promoter capable of processive sense and antisense transcription. Ovarian small RNA data show that a solo HeT-A promoter within an euchromatic transgene initiates the divergent transcription of transgenic reporter genes and subsequent processing of these transcripts into piRNAs. These events lead to the formation of a divergent unistrand piRNA cluster at solo HeT-A promoters, in contrast to endogenous telomeres that represent strong dual-strand piRNA clusters. Solo HeT-A promoters are not immunoprecipitated with heterochromatin protein 1 (HP1) homolog Rhino, a marker of the dual-strand piRNA clusters, but are associated with HP1 itself, which provides piRNA-mediated transcriptional repression of the reporter genes. Unlike endogenous dual-strand piRNA clusters, the solo HeT-A promoter does not produce overlapping transcripts. In a telomeric context, however, bidirectional promoters of tandem HeT-A repeats provide a read-through transcription of both genomic strands, followed by Rhi binding. These data indicate that Drosophila telomeres share properties of unistrand and dual-strand piRNA clusters.

RNA helicase Spn-E is required to maintain Aub and AGO3 protein levels for piRNA silencing in the germline of Drosophila.

Germline-specific RNA helicase Spindle-E (Spn-E) is known to be essential for piRNA silencing in Drosophila that takes place mainly in the perinuclear nuage granules. Loss-of-function spn-E mutations lead to tandem Stellate genes derepression in the testes and retrotransposon mobilization in the ovaries. However, Spn-E functions in the piRNA pathway are still obscure. Analysis of total library of short RNAs from the testes of spn-E heterozygous flies revealed the presence of abundant piRNA ping-pong pairs originating from Su(Ste) transcripts. The abundance of these ping-pong pairs were sharply reduced in the library from the testes of spn-E mutants. Thus we found that ping-pong mechanism contributed to Su(Ste) piRNA generation in the testes. The lack of Spn-E caused a significant drop of protein levels of key ping-pong participants, Aubergine (Aub) and AGO3 proteins of PIWI subfamily, in the germline of both males and females, but did not disrupt of their assembly in nuage granules. We found that observed decline of the protein expression was not caused by suppression of aub and ago3 transcription as well as total transcription, indicating possible contribution of Spn-E to post-transcriptional regulation.

Euchromatic transposon insertions trigger production of novel Pi- and endo-siRNAs at the target sites in the drosophila germline.

The control of transposable element (TE) activity in germ cells provides genome integrity over generations. A distinct small RNA-mediated pathway utilizing Piwi-interacting RNAs (piRNAs) suppresses TE expression in gonads of metazoans. In the fly, primary piRNAs derive from so-called piRNA clusters, which are enriched in damaged repeated sequences. These piRNAs launch a cycle of TE and piRNA cluster transcript cleavages resulting in the amplification of piRNA and TE silencing. Using genome-wide comparison of TE insertions and ovarian small RNA libraries from two Drosophila strains, we found that individual TEs inserted into euchromatic loci form novel dual-stranded piRNA clusters. Formation of the piRNA-generating loci by active individual TEs provides a more potent silencing response to the TE expansion. Like all piRNA clusters, individual TEs are also capable of triggering the production of endogenous small interfering (endo-si) RNAs. Small RNA production by individual TEs spreads into the flanking genomic regions including coding cellular genes. We show that formation of TE-associated small RNA clusters can down-regulate expression of nearby genes in ovaries. Integration of TEs into the 3' untranslated region of actively transcribed genes induces piRNA production towards the 3'-end of transcripts, causing the appearance of genic piRNA clusters, a phenomenon that has been reported in different organisms. These data suggest a significant role of TE-associated small RNAs in the evolution of regulatory networks in the germline.