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1.  Euchromatic Transposon Insertions Trigger Production of Novel Pi- and Endo-siRNAs at the Target Sites in the Drosophila Germline 
PLoS Genetics  2014;10(2):e1004138.
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.
Author Summary
Silencing of transposable elements (TEs) in germ cells depends on a distinct class of small RNAs, Piwi-interacting RNAs (piRNAs). TE repression is provided by piRNAs derived from large heterochromatic loci enriched in fragmented TE copies, so-called piRNA clusters. According to the current model, individual TEs and their transcripts are considered merely as targets of cluster-derived primary piRNAs, which exert post-transcriptional and transcriptional silencing in Drosophila. In our work, we show that natural individual transposons become piRNA-generating loci themselves. We came to this conclusion by comparing the ovarian small RNAs and TE insertion sites of two Drosophila strains, which showed that euchromatic target sites of strain-specific TEs generate a number of novel strain-specific piRNAs. This mechanism allows production of additional small RNAs that target active TEs and provide more potent transposon suppression in the germline. Moreover, small RNA production by individual TEs spreads into the flanking genomic regions, which affects the expression of adjacent coding genes and microRNA genes. These data underline the role of individual TEs in a silencing response and explore a new level of TE impact on the gene regulatory networks in the germline.
PMCID: PMC3916259  PMID: 24516406
2.  A Conserved Upstream Motif Orchestrates Autonomous, Germline-Enriched Expression of Caenorhabditis elegans piRNAs 
PLoS Genetics  2013;9(3):e1003392.
Piwi-interacting RNAs (piRNAs) fulfill a critical, conserved role in defending the genome against foreign genetic elements. In many organisms, piRNAs appear to be derived from processing of a long, polycistronic RNA precursor. Here, we establish that each Caenorhabditis elegans piRNA represents a tiny, autonomous transcriptional unit. Remarkably, the minimal C. elegans piRNA cassette requires only a 21 nucleotide (nt) piRNA sequence and an ∼50 nt upstream motif with limited genomic context for expression. Combining computational analyses with a novel, in vivo transgenic system, we demonstrate that this upstream motif is necessary for independent expression of a germline-enriched, Piwi-dependent piRNA. We further show that a single nucleotide position within this motif directs differential germline enrichment. Accordingly, over 70% of C. elegans piRNAs are selectively expressed in male or female germline, and comparison of the genes they target suggests that these two populations have evolved independently. Together, our results indicate that C. elegans piRNA upstream motifs act as independent promoters to specify which sequences are expressed as piRNAs, how abundantly they are expressed, and in what germline. As the genome encodes well over 15,000 unique piRNA sequences, our study reveals that the number of transcriptional units encoding piRNAs rivals the number of mRNA coding genes in the C. elegans genome.
Author Summary
Across the animal kingdom, Piwi-interacting small RNAs (piRNAs) protect genome integrity and promote fertility. While the functions of piRNAs are well-characterized, far less is known about how they are generated and how their expression is regulated. In the Caenorhabditis elegans genome, a conserved sequence motif lies upstream of many piRNA loci and appears to regulate their expression. We combined computational and experimental approaches to investigate the role of this motif in the expression of C. elegans piRNAs. We discovered that >70% of piRNAs are differentially enriched in male versus female germline, and these male and female piRNAs show different upstream motifs. Using a transgenic system for expressing synthetic piRNAs in vivo, we demonstrate that variation of a single nucleotide within this motif influences piRNA germline enrichment. We further show that the conserved motif is capable of driving piRNA expression in genomic isolation. Accordingly, the genomic distribution of these motifs determines which sequences are expressed as piRNAs in C. elegans. Our results suggest that each C. elegans piRNA represents an independent transcript whose sequence, abundance, and germline enrichment are encoded by a variant upstream motif, defining a novel modality for expression of piRNAs.
PMCID: PMC3597512  PMID: 23516384
3.  Drosophila Interspecific Hybrids Phenocopy piRNA-Pathway Mutants 
PLoS Biology  2012;10(11):e1001428.
Hybrids of two Drosophila species show transposable element derepression and piRNA pathway malfunction, revealing adaptive evolution of piRNA pathway components.
The Piwi-interacting RNA (piRNA) pathway defends the germline of animals from the deleterious activity of selfish transposable elements (TEs) through small-RNA mediated silencing. Adaptation to novel invasive TEs is proposed to occur by incorporating their sequences into the piRNA pool that females produce and deposit into their eggs, which then propagates immunity against specific TEs to future generations. In support of this model, the F1 offspring of crosses between strains of the same Drosophila species sometimes suffer from germline derepression of paternally inherited TE families, caused by a failure of the maternal strain to produce the piRNAs necessary for their regulation. However, many protein components of the Drosophila piRNA pathway exhibit signatures of positive selection, suggesting that they also contribute to the evolution of host genome defense. Here we investigate piRNA pathway function and TE regulation in the F1 hybrids of interspecific crosses between D. melanogaster and D. simulans and compare them with intraspecific control crosses of D. melanogaster. We confirm previous reports showing that intraspecific crosses are characterized by derepression of paternally inherited TE families that are rare or absent from the maternal genome and piRNA pool, consistent with the role of maternally deposited piRNAs in shaping TE silencing. In contrast to the intraspecific cross, we discover that interspecific hybrids are characterized by widespread derepression of both maternally and paternally inherited TE families. Furthermore, the pattern of derepression of TE families in interspecific hybrids cannot be attributed to their paucity or absence from the piRNA pool of the maternal species. Rather, we demonstrate that interspecific hybrids closely resemble piRNA effector-protein mutants in both TE misregulation and aberrant piRNA production. We suggest that TE derepression in interspecific hybrids largely reflects adaptive divergence of piRNA pathway genes rather than species-specific differences in TE-derived piRNAs.
Author Summary
Eukaryotic genomes contain large quantities of transposable elements (TEs), short self-replicating DNA sequences that can move within the genome. The selfish replication of TEs has potentially drastic consequences for the host, such as disruption of gene function, induction of sterility, and initiation or exacerbation of some cancers. Like the adaptive immune system that defends our bodies against pathogens, the Piwi-interacting RNA (piRNA) pathway defends animal genomes against the harmful effects of TEs. Fundamental to piRNA-mediated defense is the production of small noncoding RNAs that act like antibodies to target replicating TEs for destruction by piRNA-effector proteins. piRNAs are expected to diverge rapidly between species in response to genome infection by increasingly disparate TEs. Here, we tested this hypothesis by examining how differences in piRNAs between two species of fruit fly relate to TE “immunity” in their hybrid offspring. Because piRNAs are maternally deposited, we expected excessive replication of paternal TEs in hybrids. Surprisingly, we observe increased activity of both maternal and paternal TEs, together with defects in piRNA production that are reminiscent of piRNA effector-protein mutants. Our observations reveal that piRNA effector-proteins do not function properly in hybrids, and we propose that adaptive evolution among piRNA effector-proteins contributes to host genome defense and leads to the functional incompatibilities that we observe in hybrids.
PMCID: PMC3506263  PMID: 23189033
4.  proTRAC - a software for probabilistic piRNA cluster detection, visualization and analysis 
BMC Bioinformatics  2012;13:5.
Throughout the metazoan lineage, typically gonadal expressed Piwi proteins and their guiding piRNAs (~26-32nt in length) form a protective mechanism of RNA interference directed against the propagation of transposable elements (TEs). Most piRNAs are generated from genomic piRNA clusters. Annotation of experimentally obtained piRNAs from small RNA/cDNA-libraries and detection of genomic piRNA clusters are crucial for a thorough understanding of the still enigmatic piRNA pathway, especially in an evolutionary context. Currently, detection of piRNA clusters relies on bioinformatics rather than detection and sequencing of primary piRNA cluster transcripts and the stringency of the methods applied in different studies differs considerably. Additionally, not all important piRNA cluster characteristics were taken into account during bioinformatic processing. Depending on the applied method this can lead to: i) an accidentally underrepresentation of TE related piRNAs, ii) overlook duplicated clusters harboring few or no single-copy loci and iii) false positive annotation of clusters that are in fact just accumulations of multi-copy loci corresponding to frequently mapped reads, but are not transcribed to piRNA precursors.
We developed a software which detects and analyses piRNA clusters (proTRAC, probabilistic TRacking and Analysis of Clusters) based on quantifiable deviations from a hypothetical uniform distribution regarding the decisive piRNA cluster characteristics. We used piRNA sequences from human, macaque, mouse and rat to identify piRNA clusters in the respective species with proTRAC and compared the obtained results with piRNA cluster annotation from piRNABank and the results generated by different hitherto applied methods.
proTRAC identified clusters not annotated at piRNABank and rejected annotated clusters based on the absence of important features like strand asymmetry. We further show, that proTRAC detects clusters that are passed over if a minimum number of single-copy piRNA loci are required and that proTRAC assigns more sequence reads per cluster since it does not preclude frequently mapped reads from the analysis.
With proTRAC we provide a reliable tool for detection, visualization and analysis of piRNA clusters. Detected clusters are well supported by comprehensible probabilistic parameters and retain a maximum amount of information, thus overcoming the present conflict of sensitivity and specificity in piRNA cluster detection.
PMCID: PMC3293768  PMID: 22233380
5.  Human piRNAs Are Under Selection in Africans and Repress Transposable Elements 
Molecular Biology and Evolution  2011;28(11):3061-3067.
Piwi-interacting RNAs (piRNAs) are a recently discovered class of 24- to 30-nt noncoding RNAs whose best-understood function is to repress transposable elements (TEs) in animal germ lines. In humans, TE-derived sequences comprise ∼45% of the genome and there are several active TE families, including LINE-1 and Alu elements, which are a significant source of de novo mutations and intrapopulation variability. In the “ping-pong model,” piRNAs are thought to alternatively cleave sense and antisense TE transcripts in a positive feedback loop. Because piRNAs are poorly conserved between closely related species, including human and chimpanzee, we took a population genomics approach to study piRNA function and evolution. We found strong statistical evidence that piRNA sequences are under selective constraint in African populations. We then mapped the piRNA sequences to human TE sequences and found strong correlations between the age of each LINE-1 and Alu subfamily and the number of piRNAs mapping to the subfamily. This result supports the idea that piRNAs function as repressors of TEs in humans. Finally, we observed a significant depletion of piRNA matches in the reverse transcriptase region of the consensus human LINE-1 element but not of the consensus mouse LINE-1 element. This result suggests that reverse transcriptase might have an endogenous role specific to humans. Overall, our results elucidate the function and evolution of piRNAs in humans and highlight the utility of population genomics analysis for studying this rapidly evolving genetic system.
PMCID: PMC3199439  PMID: 21613236
piRNAs; transposable elements; population genetics; selective constraint; Africans
6.  Analysis of piRNA-Mediated Silencing of Active TEs in Drosophila melanogaster Suggests Limits on the Evolution of Host Genome Defense 
Molecular Biology and Evolution  2013;30(8):1816-1829.
The Piwi-interacting RNA (piRNA) pathway defends animal genomes against the harmful consequences of transposable element (TE) infection by imposing small-RNA-mediated silencing. Because silencing is targeted by TE-derived piRNAs, piRNA production is posited to be central to the evolution of genome defense. We harnessed genomic data sets from Drosophila melanogaster, including genome-wide measures of piRNA, mRNA, and genomic abundance, along with estimates of age structure and risk of ectopic recombination, to address fundamental questions about the functional and evolutionary relationships between TE families and their regulatory piRNAs. We demonstrate that mRNA transcript abundance, robustness of “ping-pong” amplification, and representation in piRNA clusters together explain the majority of variation in piRNA abundance between TE families, providing the first robust statistical support for the prevailing model of piRNA biogenesis. Intriguingly, we also discover that the most transpositionally active TE families, with the greatest capacity to induce harmful mutations or disrupt gametogenesis, are not necessarily the most abundant among piRNAs. Rather, the level of piRNA targeting is largely independent of recent transposition rate for active TE families, but is rapidly lost for inactive TEs. These observations are consistent with population genetic theory that suggests a limited selective advantage for host repression of transposition. Additionally, we find no evidence that piRNA targeting responds to selection against a second major cost of TE infection: ectopic recombination between TE insertions. Our observations confirm the pivotal role of piRNA-mediated silencing in defending the genome against selfish transposition, yet also suggest limits to the optimization of host genome defense.
PMCID: PMC3708497  PMID: 23625890
transposable elements; RNAi; genome evolution
7.  piRNAs and epigenetic conversion in Drosophila 
Fly  2013;7(4):237-241.
Transposable element (TE) activity is repressed in the Drosophila germline by Piwi-Interacting RNAs (piRNAs), a class of small non-coding RNAs. These piRNAs are produced by discrete genomic loci containing TE fragments. In a recent publication, we tested for the existence of a strict epigenetic induction of piRNA production capacity by a locus in the D. melanogaster genome. We used 2 lines carrying a transgenic 7-copy tandem cluster (P-lacZ-white) at the same genomic site. This cluster generates in both lines a local heterochromatic sector. One line (T-1) produces high levels of ovarian piRNAs homologous to the P-lacZ-white transgenes and shows a strong capacity to repress homologous sequences in trans, whereas the other line (BX2) is devoid of both of these capacities. The properties of these 2 lines are perfectly stable over generations. We have shown that the maternal transmission of a cytoplasm carrying piRNAs from the first line can confer to the inert transgenic locus of the second, a totally de novo capacity to produce high levels of piRNAs as well as the ability to induce homology-dependent silencing in trans. These new properties are stably inherited over generations (n > 50). Furthermore, the converted locus has itself become able to convert an inert transgenic locus via cytoplasmic maternal inheritance. This results in a stable epigenetic conversion process, which can be performed recurrently—a phenomenon termed paramutation and discovered in Maize 60 y ago. Paramutation in Drosophila corresponds to the first stable paramutation in animals and provides a model system to investigate the epigenetically induced emergence of a piRNA-producing locus, a crucial step in epigenome shaping. In this Extra View, we discuss some additional functional aspects and the possible molecular mechanism of this piRNA-linked paramutation.
PMCID: PMC3896495  PMID: 24088599
epigenetics; cellular memory; heterochromatin; piRNAs; transposable elements
8.  Profiles of piRNA abundances at emerging or established piRNA loci are determined by local DNA sequences 
RNA Biology  2013;10(8):1233-1239.
Piwi-interacting RNAs (piRNAs) ensure transposable element silencing in Drosophila, thereby preserving genome integrity across generations. Primary piRNAs arise from the processing of long RNA transcripts produced in the germ line by a limited number of telomeric and pericentromeric loci. Primary piRNAs bound to the Argonaute protein Aubergine then drive the production of secondary piRNAs through the “ping-pong” amplification mechanism that involves an interplay with piRNAs bound to the Argonaute protein Argonaute-3. We recently discovered that clusters of P-element-derived transgenes produce piRNAs and mediate silencing of homologous target transgenes in the female germ line. We also demonstrated that some clusters are able to convert other homologous inactive transgene clusters into piRNA-producing loci, which then transmit their acquired silencing capacity over generations. This paramutation phenomenon is mediated by maternal inheritance of piRNAs homologous to the transgenes. Here we further mined our piRNA sequencing data sets generated from various strains carrying transgenes with partial sequence homology at distinct genomic sites. This analysis revealed that same sequences in different genomic contexts generate highly similar profiles of piRNA abundances. The strong tendency of piRNAs for bearing a U at their 5′ end has long been recognized. Our observations support the notion that, in addition, the relative frequencies of Drosophila piRNAs are locally determined by the DNA sequence of piRNA loci.
PMCID: PMC3817142  PMID: 23880829
Drosophila melanogaster; argonaute proteins; epigenetics; germline; paramutation; piRNA biogenesis; transposable elements
9.  Ancient and Novel Small RNA Pathways Compensate for the Loss of piRNAs in Multiple Independent Nematode Lineages 
PLoS Biology  2015;13(2):e1002061.
Small RNA pathways act at the front line of defence against transposable elements across the Eukaryota. In animals, Piwi interacting small RNAs (piRNAs) are a crucial arm of this defence. However, the evolutionary relationships among piRNAs and other small RNA pathways targeting transposable elements are poorly resolved. To address this question we sequenced small RNAs from multiple, diverse nematode species, producing the first phylum-wide analysis of how small RNA pathways evolve. Surprisingly, despite their prominence in Caenorhabditis elegans and closely related nematodes, piRNAs are absent in all other nematode lineages. We found that there are at least two evolutionarily distinct mechanisms that compensate for the absence of piRNAs, both involving RNA-dependent RNA polymerases (RdRPs). Whilst one pathway is unique to nematodes, the second involves Dicer-dependent RNA-directed DNA methylation, hitherto unknown in animals, and bears striking similarity to transposon-control mechanisms in fungi and plants. Our results highlight the rapid, context-dependent evolution of small RNA pathways and suggest piRNAs in animals may have replaced an ancient eukaryotic RNA-dependent RNA polymerase pathway to control transposable elements.
A survey of the nematode phylum reveals loss of the Piwi/piRNA pathway in several lineages, but RNA-dependent RNA polymerases control transposable elements in its absence.
Author Summary
Transposable elements are segments of DNA that have the ability to copy themselves independently of the host genome and thus pose a severe threat to the integrity of the genome. Organisms have evolved mechanisms to restrict the spread of transposable elements, with small RNA molecules being one of the most important defense mechanisms. In animals, the predominant small RNA transposon-silencing mechanism is the piRNA pathway, which appears to be widely conserved. However, little is known about how small RNA pathways that target transposons evolve. In order to study this question we investigated small RNA pathways across the nematode phylum, using a well-studied model organism—the nematode Caenorhabditis elegans—as the starting point. Surprisingly we found that the piRNA pathway has been completely lost in all groups of nematodes bar those most closely related to C. elegans. This finding raises the intriguing question of how these nematodes are able to control transposable element mobilization without piRNAs. We discovered that there are other small RNA pathways that target transposable elements in these nematodes, employing RNA-dependent RNA polymerases in order to make small RNAs antisense to transposable elements. Intriguingly, the most ancient of these mechanisms, found in the most basal nematodes, is a Dicer-dependent RNA-directed DNA methylation pathway. This pathway shares strong similarity to transposon-silencing mechanisms in plants and fungi, suggesting that it might have been present in an ancient common ancestor of all eukaryotes. Our results highlight the rapid evolution of small RNA pathways and demonstrate the importance of examining molecular pathways in detail across a range of evolutionary distances.
PMCID: PMC4323106  PMID: 25668728
10.  Computational Analysis of Mouse piRNA Sequence and Biogenesis 
PLoS Computational Biology  2007;3(11):e222.
The recent discovery of a new class of 30-nucleotide long RNAs in mammalian testes, called PIWI-interacting RNA (piRNA), with similarities to microRNAs and repeat-associated small interfering RNAs (rasiRNAs), has raised puzzling questions regarding their biogenesis and function. We report a comparative analysis of currently available piRNA sequence data from the pachytene stage of mouse spermatogenesis that sheds light on their sequence diversity and mechanism of biogenesis. We conclude that (i) there are at least four times as many piRNAs in mouse testes than currently known; (ii) piRNAs, which originate from long precursor transcripts, are generated by quasi-random enzymatic processing that is guided by a weak sequence signature at the piRNA 5′ends resulting in a large number of distinct sequences; and (iii) many of the piRNA clusters contain inverted repeats segments capable of forming double-strand RNA fold-back segments that may initiate piRNA processing analogous to transposon silencing.
Author Summary
The discovery of a new class of mammalian small regulatory RNAs termed PIWI-interacting RNA (piRNA) has extended the diverse family of small regulatory RNAs. PIWI proteins are a subclass of the larger Argonaute proteins family, of which the Ago members bind microRNAs and play a critical role in gene silencing. Despite the homology between PIWI and Ago proteins, piRNAs are strikingly different from microRNAs in their length, expression pattern, and genomic organization. In contrast, piRNAs are similar to repeat-associated small interfering RNA (rasiRNAs), a class of small RNAs that are responsible for transposon silencing in Drosophila germline, although it is unclear if piRNAs function in a similar way. This paper describes a computational comparison and analysis of the existing comprehensive piRNA datasets identified independently by three groups at the pachytene stage in mouse spermatogenesis. We find that the studies have identified similar genomic piRNA clusters, but differ substantially in the piRNAs that were cloned from those clusters. Based on these results we quantify the expected number of piRNAs and suggest that the processing of piRNAs from genomic transcripts is quasi-random. We find that a weak sequence signature may guide the piRNA 5′end processing that accounts for the departure from fully random processing. We further show partial evidence that piRNA biogenesis may be initiated by neighboring transposable elements.
PMCID: PMC2065894  PMID: 17997596
11.  A comprehensive analysis of piRNAs from adult human testis and their relationship with genes and mobile elements 
BMC Genomics  2014;15(1):545.
Piwi-interacting RNAs (piRNAs) are a recently discovered class of small non-coding RNAs whose best-understood function is to repress mobile element (ME) activity in animal germline. To date, nearly all piRNA studies have been conducted in model organisms and little is known about piRNA diversity, target specificity and biological function in human.
Here we performed high-throughput sequencing of piRNAs from three human adult testis samples. We found that more than 81% of the ~17 million putative piRNAs mapped to ~6,000 piRNA-producing genomic clusters using a relaxed definition of clusters. A set of human protein-coding genes produces a relatively large amount of putative piRNAs from their 3’UTRs, and are significantly enriched for certain biological processes, suggestive of non-random sampling by the piRNA biogenesis machinery. Up to 16% of putative piRNAs mapped to a few hundred annotated long non-coding RNA (lncRNA) genes, suggesting that some lncRNA genes can act as piRNA precursors. Among major ME families, young families of LTR and endogenous retroviruses have a greater association with putative piRNAs than other MEs. In addition, piRNAs preferentially mapped to specific regions in the consensus sequences of several ME (sub)families and some piRNA mapping peaks showed patterns consistent with the “ping-pong” cycle of piRNA targeting and amplification.
Overall our data provide a comprehensive analysis and improved annotation of human piRNAs in adult human testes and shed new light into the relationship of piRNAs with protein-coding genes, lncRNAs, and mobile genetic elements in human.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-545) contains supplementary material, which is available to authorized users.
PMCID: PMC4094622  PMID: 24981367
Human piRNA; piRNA cluster; Protein coding gene; Mobile element; High-throughput sequencing
12.  Rapid Evolution of piRNA Pathway in the Teleost Fish: Implication for an Adaptation to Transposon Diversity 
Genome Biology and Evolution  2014;6(6):1393-1407.
The Piwi-interacting RNA (piRNA) pathway is responsible for germline specification, gametogenesis, transposon silencing, and genome integrity. Transposable elements can disrupt genome and its functions. However, piRNA pathway evolution and its adaptation to transposon diversity in the teleost fish remain unknown. This article unveils evolutionary scene of piRNA pathway and its association with diverse transposons by systematically comparative analysis on diverse teleost fish genomes. Selective pressure analysis on piRNA pathway and miRNA/siRNA (microRNA/small interfering RNA) pathway genes between teleosts and mammals showed an accelerated evolution of piRNA pathway genes in the teleost lineages, and positive selection on functional PAZ (Piwi/Ago/Zwille) and Tudor domains involved in the Piwi–piRNA/Tudor interaction, suggesting that the amino acid substitutions are adaptive to their functions in piRNA pathway in the teleost fish species. Notably five piRNA pathway genes evolved faster in the swamp eel, a kind of protogynous hermaphrodite fish, than the other teleosts, indicating a differential evolution of piRNA pathway between the swamp eel and other gonochoristic fishes. In addition, genome-wide analysis showed higher diversity of transposons in the teleost fish species compared with mammals. Our results suggest that rapidly evolved piRNA pathway in the teleost fish is likely to be involved in the adaption to transposon diversity.
PMCID: PMC4079211  PMID: 24846630
teleost fish; evolution; positive selection; reproduction
13.  A broadly conserved pathway generates 3' UTR-directed primary piRNAs 
Current biology : CB  2009;19(24):2066-2076.
Piwi-interacting RNAs (piRNAs) are ~24–30 nucleotide regulatory RNAs that are abundant in animal gonads and early embryos. The best characterized piRNAs mediate a conserved pathway that restricts transposable elements, and these frequently engage a "ping-pong" amplification loop. Certain stages of mammalian testis also accumulate abundant piRNAs of unknown function, which derive from non-coding RNAs that are depleted in TE content and do not engage in ping-pong.
We report that the 3' untranslated regions (3' UTRs) of an extensive set of messenger RNAs (mRNAs) are processed into piRNAs in Drosophila ovaries, murine testes, and Xenopus eggs. Analysis of small RNA data from different mutants and Piwi-class immunoprecipitates indicates that their biogenesis depends on primary piRNA components but not ping-pong components. Several observations suggest that mRNAs are actively selected for piRNA production. First, genic piRNAs do not accumulate in proportion to the level of their host transcripts, and many highly expressed transcripts lack piRNAs. Second, piRNA-producing mRNAs in Drosophila and mouse are enriched for specific gene ontology categories distinct from those of simply abundant transcripts. Third, the levels of Traffic Jam, whose 3' UTR generates abundant piRNAs, are increased in piwi mutant follicle clones. These data suggest that selection of cellular transcripts by the primary piRNA pathway is not fortuitous, but instead an active process with regulatory consequences.
Our work reveals a conserved primary piRNA pathway that selects and metabolizes the 3' UTRs of a broad set of cellular transcripts, providing insights into piRNA biogenesis and function. These data strongly increase the breadth of Argonaute-mediated small RNA systems in metazoans.
PMCID: PMC2812478  PMID: 20022248
14.  The Cutoff protein regulates piRNA cluster expression and piRNA production in the Drosophila germline 
The EMBO Journal  2011;30(22):4601-4615.
The Cutoff protein regulates piRNA cluster expression and piRNA production in the Drosophila germline
The identity and function of many factors involved in the piRNA pathway remain unknown. Here, in Drosophila, cutoff plays a role in regulating piRNA cluster transcript levels and biogenesis together with the heterochromatin protein Rhino.
In a broad range of organisms, Piwi-interacting RNAs (piRNAs) have emerged as core components of a surveillance system that protects the genome by silencing transposable and repetitive elements. A vast proportion of piRNAs is produced from discrete genomic loci, termed piRNA clusters, which are generally embedded in heterochromatic regions. The molecular mechanisms and the factors that govern their expression are largely unknown. Here, we show that Cutoff (Cuff), a Drosophila protein related to the yeast transcription termination factor Rai1, is essential for piRNA production in germline tissues. Cuff accumulates at centromeric/pericentromeric positions in germ-cell nuclei and strongly colocalizes with the major heterochromatic domains. Remarkably, we show that Cuff is enriched at the dual-strand piRNA cluster 1/42AB and is likely to be involved in regulation of transcript levels of similar loci dispersed in the genome. Consistent with this observation, Cuff physically interacts with the Heterochromatin Protein 1 (HP1) variant Rhino (Rhi). Our results unveil a link between Cuff activity, heterochromatin assembly and piRNA cluster expression, which is critical for stem-cell and germ-cell development in Drosophila.
PMCID: PMC3243597  PMID: 21952049
cutoff; Drosophila; germline; heterochromatin; piRNA
15.  Blockade of Pachytene piRNA Biogenesis Reveals a Novel Requirement for Maintaining Post-Meiotic Germline Genome Integrity 
PLoS Genetics  2012;8(11):e1003038.
Piwi-interacting RNAs are a diverse class of small non-coding RNAs implicated in the silencing of transposable elements and the safeguarding of genome integrity. In mammals, male germ cells express two genetically and developmentally distinct populations of piRNAs at the pre-pachytene and pachytene stages of meiosis, respectively. Pre-pachytene piRNAs are mostly derived from retrotransposons and required for their silencing. In contrast, pachytene piRNAs originate from ∼3,000 genomic clusters, and their biogenesis and function remain enigmatic. Here, we report that conditional inactivation of the putative RNA helicase MOV10L1 in mouse spermatocytes produces a specific loss of pachytene piRNAs, significant accumulation of pachytene piRNA precursor transcripts, and unusual polar conglomeration of Piwi proteins with mitochondria. Pachytene piRNA–deficient spermatocytes progress through meiosis without derepression of LINE1 retrotransposons, but become arrested at the post-meiotic round spermatid stage with massive DNA damage. Our results demonstrate that MOV10L1 acts upstream of Piwi proteins in the primary processing of pachytene piRNAs and suggest that, distinct from pre-pachytene piRNAs, pachytene piRNAs fulfill a unique function in maintaining post-meiotic genome integrity.
Author Summary
Small non-coding RNAs play critical roles during development and in disease. The integrity of the germline genome is of paramount importance to the wellbeing of offspring and the survival of species. Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs abundantly expressed in the gonad. Compared to microRNAs and small-interfering RNAs (siRNAs), the biogenesis and function of piRNAs remain poorly understood. Here we have identified MOV10L1, a putative RNA helicase, as a master regulator of piRNA biogenesis in mouse. We find that production of pachytene piRNAs requires MOV10L1. Blockade of pachytene piRNAs disrupts germ cell development and results in defects in post-meiotic genome integrity. Therefore, mutations in MOV10L1 and other piRNA pathway components may contribute to male infertility in humans.
PMCID: PMC3499362  PMID: 23166510
16.  Cytoplasmic Compartmentalization of the Fetal piRNA Pathway in Mice 
PLoS Genetics  2009;5(12):e1000764.
Derepression of transposable elements (TEs) in the course of epigenetic reprogramming of the mouse embryonic germline necessitates the existence of a robust defense that is comprised of PIWI/piRNA pathway and de novo DNA methylation machinery. To gain further insight into biogenesis and function of piRNAs, we studied the intracellular localization of piRNA pathway components and used the combination of genetic, molecular, and cell biological approaches to examine the performance of the piRNA pathway in germ cells of mice lacking Maelstrom (MAEL), an evolutionarily conserved protein implicated in transposon silencing in fruit flies and mice. Here we show that principal components of the fetal piRNA pathway, MILI and MIWI2 proteins, localize to two distinct types of germinal cytoplasmic granules and exhibit differential association with components of the mRNA degradation/translational repression machinery. The first type of granules, pi-bodies, contains the MILI-TDRD1 module of the piRNA pathway and is likely equivalent to the enigmatic “cementing material” first described in electron micrographs of rat gonocytes over 35 years ago. The second type of granules, piP-bodies, harbors the MIWI2-TDRD9-MAEL module of the piRNA pathway and signature components of P-bodies, GW182, DCP1a, DDX6/p54, and XRN1 proteins. piP-bodies are found predominantly in the proximity of pi-bodies and the two frequently share mouse VASA homolog (MVH) protein, an RNA helicase. In Mael-mutant gonocytes, MIWI2, TDRD9, and MVH are lost from piP-bodies, whereas no effects on pi-body composition are observed. Further analysis revealed that MAEL appears to specifically facilitate MIWI2-dependent aspects of the piRNA pathway including biogenesis of secondary piRNAs, de novo DNA methylation, and efficient downregulation of TEs. Cumulatively, our data reveal elaborate cytoplasmic compartmentalization of the fetal piRNA pathway that relies on MAEL function.
Author Summary
Vast territories of animal genomes are populated by numerous types of mobile genetic elements (or transposons) that act predominantly as selfish parasites unconcerned with the impact of their activity on the well-being of the host. In response to the danger posed by transposons, organisms have evolved a defensive mechanism that employs a particular class of small RNAs known as piRNAs to identify and selectively silence transposons. We have studied the subcellular organization of such a defensive mechanism, the piRNA pathway, in germ cells of mouse male embryos. We discovered that key proteins involved in the genesis of small RNAs, MILI and MIWI2, occupy specific domains within the cytoplasm of germ cells. Surprisingly, MIWI2 shares its domain with proteins known to degrade RNAs and repress synthesis of cellular proteins, thus raising a possibility of cooperation of the two mechanisms in transposon defense. Genetic ablation of MAEL, a protein also found within the MIWI2 domain, disrupts normal MIWI2 localization and piRNA production leading to transposon activation. This study demonstrates that an elaborate compartmentalization of the defensive mechanism is required for the efficient recognition and destruction of active transposons in germ cells of mice.
PMCID: PMC2785470  PMID: 20011505
17.  The distribution of a germline methylation marker suggests a regional mechanism of LINE-1 silencing by the piRNA-PIWI system 
BMC Genetics  2012;13:31.
A defense system against transposon activity in the human germline based on PIWI proteins and piRNA has recently been discovered. It represses the activity of LINE-1 elements via DNA methylation by a largely unknown mechanism. Based on the dispersed distribution of clusters of piRNA genes in a strand-specific manner on all human chromosomes, we hypothesized that this system might work preferentially on local and proximal sequences. We tested this hypothesis with a methylation-associated SNP (mSNP) marker which is based on the density of C-T transitions in CpG dinucleotides as a surrogate marker for germline methylation.
We found significantly higher density of mSNPs flanking piRNA clusters in the human genome for flank sizes of 1-16 Mb. A dose-response relationship between number of piRNA genes and mSNP density was found for up to 16 Mb of flanking sequences. The chromosomal density of hypermethylated LINE-1 elements had a significant positive correlation with the chromosomal density of piRNA genes (r = 0.41, P = 0.05). Genome windows of 1-16 Mb containing piRNA clusters had significantly more hypermethylated LINE-1 elements than windows not containing piRNA clusters. Finally, the minimum distance to the next piRNA cluster was significantly shorter for hypermethylated LINE-1 compared to normally methylated elements (14.4 Mb vs 16.1 Mb).
Our observations support our hypothesis that the piRNA-PIWI system preferentially methylates sequences in close proximity to the piRNA clusters and perhaps physically adjacent sequences on other chromosomes. Furthermore they suggest that this proximity effect extends up to 16 Mb. This could be due to an unknown localization signal, transcription of piRNA genes near the nuclear membrane or the presence of an unknown RNA molecule that spreads across the chromosome and targets the methylation directed by the piRNA-PIWI complex. Our data suggest a region specific molecular mechanism which can be sought experimentally.
PMCID: PMC3352111  PMID: 22530917
piRNA; PIWI; LINE-1; Transposable element; Germline; Methylation; Epigenetics
18.  HP1 Recruitment in the Absence of Argonaute Proteins in Drosophila 
PLoS Genetics  2010;6(3):e1000880.
Highly repetitive and transposable element rich regions of the genome must be stabilized by the presence of heterochromatin. A direct role for RNA interference in the establishment of heterochromatin has been demonstrated in fission yeast. In metazoans, which possess multiple RNA–silencing pathways that are both functionally distinct and spatially restricted, whether RNA silencing contributes directly to heterochromatin formation is not clear. Previous studies in Drosophila melanogaster have suggested the involvement of both the AGO2-dependent endogenous small interfering RNA (endo-siRNA) as well as Piwi-interacting RNA (piRNA) silencing pathways. In order to determine if these Argonaute genes are required for heterochromatin formation, we utilized transcriptional reporters and chromatin immunoprecipitation of the critical factor Heterochromatin Protein 1 (HP1) to monitor the heterochromatic state of piRNA clusters, which generate both endo-siRNAs and the bulk of piRNAs. Surprisingly, we find that mutation of AGO2 or piwi increases silencing at piRNA clusters corresponding to an increase of HP1 association. Furthermore, loss of piRNA production from a single piRNA cluster results in genome-wide redistribution of HP1 and reduction of silencing at a distant heterochromatic site, suggesting indirect effects on HP1 recruitment. Taken together, these results indicate that heterochromatin forms independently of endo-siRNA and piRNA pathways.
Author Summary
One role for silent heterochromatin is to preserve the integrity of the genome by stabilizing regions rich in repetitive sequence and mobile elements. Compaction of repetitive sequences by heterochromatin is needed to prevent genome rearrangement and loss of genetic material. Furthermore, uncontrolled movement of mobile elements throughout the genome can result in deleterious mutations. In fission yeast, one important mechanism of heterochromatin establishment occurs through RNA interference, an RNA–dependent gene silencing process. However, it is unclear whether a direct role for RNA silencing in heterochromatin formation is conserved throughout evolution. In the fruit fly, Drosophila melanogaster, which harbors multiple RNA–silencing pathways that are both functionally distinct and spatially restricted, previous studies have suggested the involvement of the endogenous small interfering RNA (endo-siRNA) and Piwi-interacting RNA (piRNA) pathways in heterochromatin formation. These small RNA silencing pathways suppress the expression of mobile elements in the soma or in both somatic and germline tissues, respectively. Utilizing complementary genetic and biochemical approaches, we monitored the heterochromatin state at discrete genomic locations from which both types of these small RNAs originate in endo-siRNA or piRNA pathway mutants. Our results indicate that heterochromatin can form independently of these two small RNA silencing pathways.
PMCID: PMC2837403  PMID: 20300658
19.  Function, targets and evolution of Caenorhabditis elegans piRNAs 
Science (New York, N.Y.)  2012;337(6094):574-578.
Piwi-interacting RNAs (piRNAs) are small RNAs required to maintain germline integrity and fertility but their mechanism of action is poorly understood. Here we demonstrate that C. elegans piRNAs silence transcripts in trans through imperfectly complementary sites. Target silencing is independent of Piwi endonuclease activity or “slicing”. Instead, piRNAs initiate a localized secondary endogenous small interfering RNA (endo-siRNA) response. Endogenous protein-coding gene and transposon transcripts exhibit Piwi-dependent endo-siRNAs at sites complementary to piRNAs and are de-repressed in Piwi mutants. Genomic loci of piRNA biogenesis are depleted of protein-coding genes and tend to overlap the start and end of transposons in sense and antisense, respectively. Our data suggest that nematode piRNA clusters are evolving to generate piRNAs against active mobile elements. Thus, piRNAs provide heritable, sequence-specific triggers for RNAi in C. elegans.
PMCID: PMC3951736  PMID: 22700655
20.  The Tudor domain protein Tapas, a homolog of the vertebrate Tdrd7, functions in the piRNA pathway to regulate retrotransposons in germline of Drosophila melanogaster 
BMC Biology  2014;12(1):61.
Piwi-interacting RNAs (piRNAs) are a special class of small RNAs that provide defense against transposable elements in animal germline cells. In Drosophila, germline piRNAs are thought to be processed at a unique perinuclear structure, the nuage, that houses piRNA pathway proteins including the Piwi clade of Argonaute family proteins, along with several Tudor domain proteins, RNA helicases and nucleases. We previously demonstrated that Tudor domain protein Tejas (Tej), an ortholog of vertebrate Tdrd5, is an important component of the piRNA pathway.
In the current study, we identified the paralog of the Drosophila tej gene, tapas (tap), which is an ortholog of vertebrate Tdrd7. Like Tej, Tap is localized at the nuage. Alone, tap loss leads to a mild increase in transposon expression and decrease in piRNAs targeting transposons expressed in the germline. The tap gene genetically interacts with other piRNA pathway genes and we also show that Tap physically interacts with piRNA pathway components, such as Piwi family proteins Aubergine and Argonaute3 and the RNA helicases Spindle-E and Vasa. Together with tej, tap is required for survival of germline cells during early stages and for polarity formation. We further observed that loss of tej and tap together results in more severe defects in the piRNA pathway in germline cells compared to single mutants: the double-mutant ovaries exhibit mis-localization of piRNA pathway components and significantly greater reduction of piRNAs against transposons predominantly expressed in germline compared to single mutants. The single or double mutants did not have any reduction in piRNAs mapping to transposons predominantly expressed in gonadal somatic cells or those derived from unidirectional clusters such as flamenco. Consistently, the loss of both tej and tap function resulted in mis-localization of Piwi in germline cells, whereas Piwi remained localized to the nucleus in somatic cells.
Our observations suggest that tej and tap work together for germline maintenance. tej and tap also function in a synergistic manner to maintain examined piRNA components at the perinuclear nuage and for piRNA production in Drosophila germline cells.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-014-0061-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4210518  PMID: 25287931
Germline; Nuage; piRNA; Tudor domain
21.  Retrotransposon-centered analysis of piRNA targeting shows a shift from active to passive retrotransposon transcription in developing mouse testes 
BMC Genomics  2011;12:440.
Piwi-associated RNAs (piRNAs) bind transcripts from retrotransposable elements (RTE) in mouse germline cells and seemingly act as guides for genomic methylation, thereby repressing the activity of RTEs. It is currently unknown if and how Piwi proteins distinguish RTE transcripts from other cellular RNAs. During germline development, the main target of piRNAs switch between different types of RTEs. Using the piRNA targeting of RTEs as an indicator of RTE activity, and considering the entire population of genomic RTE loci along with their age and location, this study aims at further elucidating the dynamics of RTE activity during mouse germline development.
Due to the inherent sequence redundancy between RTE loci, assigning piRNA targeting to specific loci is problematic. This limits the analysis, although certain features of piRNA targeting of RTE loci are apparent. As expected, young RTEs display a much higher level of piRNA targeting than old RTEs. Further, irrespective of age, RTE loci near protein-coding coding genes are targeted to a greater extent than RTE loci far from genes. During development, a shift in piRNA targeting is observed, with a clear increase in the relative piRNA targeting of RTEs residing within boundaries of protein-coding gene transcripts.
Reanalyzing published piRNA sequences and taking into account the features of individual RTE loci provide novel insight into the activity of RTEs during development. The obtained results are consistent with some degree of proportionality between what transcripts become substrates for Piwi protein complexes and the level by which the transcripts are present in the cell. A transition from active transcription of RTEs to passive co-transcription of RTE sequences residing within protein-coding transcripts appears to take place in postnatal development. Hence, the previously reported increase in piRNA targeting of SINEs in postnatal testis development does not necessitate widespread active transcription of SINEs, but may simply be explained by the prevalence of SINEs residing in introns.
PMCID: PMC3175481  PMID: 21884594
22.  Discovery of Potential piRNAs from Next Generation Sequences of the Sexually Mature Porcine Testes 
PLoS ONE  2012;7(4):e34770.
Piwi- interacting RNAs (piRNAs), a new class of small RNAs discovered from mammalian testes, are involved in transcriptional silencing of retrotransposons and other genetic elements in germ line cells. In order to identify a full transcriptome set of piRNAs expressed in the sexually mature porcine testes, small RNA fractions were extracted and were subjected to a Solexa deep sequencing. We cloned 6,913,561 clean reads of Sus Scrofa small RNAs (18–30 nt) and performed functional characterization. Sus Scrofa small RNAs showed a bimodal length distribution with two peaks at 21 nt and 29 nt. Then from 938,328 deep-sequenced small RNAs (26–30 nt), 375,195 piRNAs were identified by a k-mer scheme and 326 piRNAs were identified by homology searches. All piRNAs predicted by the k-mer scheme were then mapped to swine genome by Short Oligonucleotide Analysis Package (SOAP), and 81.61% of all uniquely mapping piRNAs (197,673) were located to 1124 defined genomic regions (5.85 Mb). Within these regions, 536 and 501 piRNA clusters generally distributed across only minus or plus genomic strand, 48 piRNA clusters distributed on two strands but in a divergent manner, and 39 piRNA clusters distributed on two strands in an overlapping manner. Furthermore, expression pattern of 7 piRNAs identified by homology searches showed 5 piRNAs displayed a ubiquitous expression pattern, although 2 piRNAs were specifically expressed in the testes. Overall, our results provide new information of porcine piRNAs and their specific expression pattern in porcine testes suggests that piRNAs have a role in regulating spermatogenesis.
PMCID: PMC3321025  PMID: 22493715
23.  piRNA clusters and open chromatin structure 
Mobile DNA  2014;5:22.
Transposable elements (TEs) are major structural components of eukaryotic genomes; however, mobilization of TEs generally has negative effects on the host genome. To counteract this threat, host cells have evolved genetic and epigenetic mechanisms that keep TEs silenced. One such mechanism involves the Piwi-piRNA complex, which represses TEs in animal gonads either by cleaving TE transcripts in the cytoplasm or by directing specific chromatin modifications at TE loci in the nucleus. Most Piwi-interacting RNAs (piRNAs) are derived from genomic piRNA clusters. There has been remarkable progress in our understanding of the mechanisms underlying piRNA biogenesis. However, little is known about how a specific locus in the genome is converted into a piRNA-producing site. In this review, we will discuss a possible link between chromatin boundaries and piRNA cluster formation.
PMCID: PMC4131230  PMID: 25126116
Transposable elements; Piwi; piRNA; piRNA cluster; Chromatin boundary
24.  Endogenous RNA Interference Provides a Somatic Defense against Drosophila Transposons 
Current biology : CB  2008;18(11):795-802.
Because of the mutagenic consequences of mobile genetic elements, elaborate defenses have evolved to restrict their activity. A major system that controls the activity of transposable elements (TEs) in flies and vertebrates is mediated by Piwi-interacting RNAs (piRNAs), which are ~24–30 nucleotide RNAs that are bound by Piwi-class effectors. The piRNA system is thought to provide primarily a germline defense against TE activity.
Here, we describe a second system that represses Drosophila TEs by using endogenous small interfering RNAs (si RNAs), which are 21 nucleotide, 3′-end-modified RNAs that are dependent on Dicer-2 and Argonaute-2. In contrast to piRNAs, we find that the TE-siRNA system is active in somatic tissues, and particularly so in various immortalized cell lines. Analysis of the patterns and properties of TE-derived small RNAs reveals further distinctions between TE regions and genomic loci that are converted into piRNAs and siRNAs, respectively. Finally, functional tests show that many transposon transcripts accumulate to higher levels in cells and animal tissues that are deficient for Dicer-2 or Argonaute-2.
Drosophila utilizes two small-RNA systems to restrict transposon activity in the germline (mostly via piRNAs) and in the soma (mostly via siRNAs).
PMCID: PMC2812477  PMID: 18501606
25.  Variation in piRNA and Transposable Element Content in Strains of Drosophila melanogaster 
Genome Biology and Evolution  2014;6(10):2786-2798.
Transposable elements (TEs) are one of the most important features of genome architecture, so their evolution and relationship with host defense mechanisms have been topics of intense study, especially in model systems such as Drosophila melanogaster. Recently, a novel small RNA-based defense mechanism in animals called the Piwi-interacting RNA (piRNA) pathway was discovered to form an adaptive defense mechanism against TEs. To investigate the relationship between piRNA and TE content between strains of a species, we sequenced piRNAs from 16 inbred lines of D. melanogaster from the Drosophila Genetic Reference Panel. Instead of a global correlation of piRNA expression and TE content, we found evidence for a host response through de novo piRNA production from novel TE insertions. Although approximately 20% of novel TE insertions induced de novo piRNA production, the abundance of de novo piRNAs was low and did not markedly affect the global pool of ovarian piRNAs. Our results provide new insights into the evolution of TEs and the piRNA system in an important model organism.
PMCID: PMC4224344  PMID: 25267446
piRNA; transposable elements; Drosophila melanogaster; de novo piRNA production

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