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.
piRNAs; transposable elements; population genetics; selective constraint; Africans
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.
cutoff; Drosophila; germline; heterochromatin; piRNA
Piwi proteins, together with their bound Piwi-interacting RNAs, constitute an evolutionarily conserved, germline-specific innate immune system. The piRNA pathway is one of the key mechanisms for silencing transposable elements in the germline, thereby preserving genome integrity between generations. Recent work from several groups has significantly advanced our understanding of how piRNAs arise from discrete genomic loci, termed piRNA clusters, and how these Piwi-piRNA complexes enforce transposon silencing. Here, we discuss these recent findings, as well as highlight some aspects of piRNA biology that continue to escape our understanding.
Small non-coding RNAs have emerged as key players in epigenetic regulation. Recently, a novel class of small RNAs that interact with Piwi proteins has been discovered in the mammalian and Drosophila germline. These Piwi-interacting RNAs (piRNAs) represent a distinct small RNA pathway that is widely thought to function only in the germline. In this essay, we review our recent work with our collaborators on the epigenetic function of the Drosophila Piwi protein and its associated piRNAs in somatic cells. This work has revealed a novel epigenetic mechanism mediated by Piwi and its associated piRNAs in somatic cells that might also be applicable to the germline. Based on these results, we propose a “Piwi-piRNA guidance hypothesis” for Piwi/piRNA-mediated epigenetic programming, in which the Piwi-piRNA complex serves as a sequence-recognition machinery that recruits epigenetic effectors such as Heterochromatin Protein 1a (HP1a) to specific sites in the genome to execute epigenetic regulation.
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.
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.
Piwi-interacting RNA (piRNA) are small RNA abundant in the germline across animal species. In fruit flies and mice, piRNA have been implicated in maintenance of genomic integrity by transposable elements silencing. Outside of the germline, piRNA have only been found in fruit fly ovarian follicle cells. Previous studies have further reported presence of multiple piRNA-like small RNA (pilRNA) in fly heads and a small number of pilRNA have been reported in mouse tissues and in human NK cells. Here, we analyze high-throughput small RNA sequencing data in more than 130 fruit fly, mouse and rhesus macaque samples. The results show widespread presence of pilRNA, displaying all known characteristics of piRNA in multiple somatic tissues of these three species. In mouse pancreas and macaque epididymis, pilRNA abundance was compatible with piRNA abundance in the germline. Using in situ hybridizations, we further demonstrate pilRNA co-localization with mRNA expression of Piwi-family genes in all macaque tissues. Further, using western blot, we have shown the expression of Miwi protein in mouse pancreas. These findings indicate that piRNA-like molecules might play important roles outside of the germline.
Piwi-interacting RNAs (piRNAs) and CRISPR RNAs (crRNAs) are two recently discovered classes of small noncoding RNA that are found in animals and prokaryotes, respectively. Both of these novel RNA species function as components of adaptive immune systems that protect their hosts from foreign nucleic acids—piRNAs repress transposable elements in animal germlines, whereas crRNAs protect their bacterial hosts from phage and plasmids. The piRNA and CRISPR systems are nonhomologous but rather have independently evolved into logically similar defense mechanisms based on the specificity of targeting via nucleic acid base complementarity. Here we review what is known about the piRNA and CRISPR systems with a focus on comparing their evolutionary properties. In particular, we highlight the importance of several factors on the pattern of piRNA and CRISPR evolution, including the population genetic environment, the role of alternate defense systems and the mechanisms of acquisition of new piRNAs and CRISPRs.
piRNA; CRISPR; co-evolution; transposable elements; phage; plasmids
Piwi Argonautes and Piwi-interacting RNAs (piRNAs) mediate genome defense by targeting transposons. However, many piRNA species lack obvious sequence complementarity to transposons or other loci; only one C. elegans transposon is a known piRNA target. Here we show that, in mutants lacking the Piwi Argonaute PRG-1 (and consequently its associated piRNAs/21U-RNAs), many silent loci in the germline exhibit increased levels of mRNA expression and depletion of an amplified RNA-dependent RNA polymerase (RdRP)-derived species of small secondary RNA termed 22G-RNAs. Sequences depleted of 22G-RNAs are enriched at nearby potential target sites that base pair imperfectly but extensively to 21U-RNAs. We show that PRG-1 is required to initiate, but not to maintain, silencing of transgenes engineered to contain complementarity to endogenous 21U-RNAs. Our findings support a model in which C. elegans piRNAs utilize their enormous repertoire of targeting capacity to scan the germline transcriptome for foreign sequences, while endogenous germline-expressed genes are actively protected from piRNA-induced silencing.
Piwi-interacting RNAs (piRNAs) silence transposons in the germ line of animals. They are thought to derive from long primary transcripts spanning transposon-rich genomic loci, “piRNA clusters.” piRNAs are proposed to direct an auto-amplification loop in which an antisense piRNA, bound to Aubergine or Piwi protein, directs the cleavage of sense RNA, triggering production of a sense piRNA bound to the PIWI protein Argonaute3 (Ago3). In turn, the new piRNA is envisioned to direct cleavage of a cluster transcript, initiating production of a second antisense piRNA. Here, we describe strong loss-of-function mutations in ago3, allowing a direct genetic test of this model. We find that Ago3 acts to amplify piRNA pools and to enforce on them an antisense bias, increasing the number of piRNAs that can act to silence transposons. We also detect a second piRNA pathway centered on Piwi and functioning without benefit of Ago3-catalyzed amplification. Transposons targeted by this second pathway often reside in the flamenco locus, which is expressed in somatic ovarian follicle cells, suggesting a role for piRNAs beyond the germ line.
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).
Piwi interacting RNA, or piRNA, is a class of small RNA almost exclusively expressed in the germline where they serve essential roles in retrotransposon silencing. There are two types, primary and secondary piRNA, and the latter is a product of enzymatic cleavage of retrotransposons' transcripts directed by the former. Recently, a new class of 19nt long RNA was discovered that is specific to testis and appears to be linked to secondary piRNA biogenesis.
We locate clusters of the testis-specific 19mers, which we call piRNA-related 19mers (pr19RNA), and characterise the transcripts from which they are derived. Most pr19RNA clusters were associated with retrotransposons and unannotated antisense transcripts overlapping piRNA clusters. At these loci the abundance of 19mers was found to be greater than that of secondary piRNAs.
We find that pr19RNAs are distinguished from other RNA populations by their length and flanking sequence, allowing their identification without requiring overlapping piRNAs. Using such sequence features allows identification of the source transcripts, and we suggest that these likely represent the substrates of primary piRNA-guided RNA cleavage events. While pr19RNAs appear not to bind directly to Miwi or Mili, their abundance relative to secondary piRNAs, in combination with their precise length, suggests they may be more than by-products of secondary piRNA biogenesis.
PIWI-interacting RNAs (piRNAs) are germline-specific small non-coding RNAs that form piRNA-induced silencing complexes (piRISCs) by associating with PIWI proteins, a subclade of the Argonaute proteins predominantly expressed in the germline. piRISCs protect the integrity of the germline genome from invasive transposable DNA elements by silencing them. Multiple piRNA biogenesis factors have been identified in Drosophila. The majority of piRNA factors are localized in the nuage, electron-dense non-membranous cytoplasmic structures located in the perinuclear regions of germ cells. Thus, piRNA biogenesis is thought to occur in the nuage in germ cells. Immunofluorescence analyses of ovaries from piRNA factor mutants have revealed a localization hierarchy of piRNA factors in female nuage. However, whether this hierarchy is female-specific or can also be applied in male gonads remains undetermined. Here, we show by immunostaining of both ovaries and testes from piRNA factor mutants that the molecular hierarchy of piRNA factors shows gender-specificity, especially for Krimper (Krimp), a Tudor-domain-containing protein of unknown function(s): Krimp is dispensable for PIWI protein Aubergine (Aub) nuage localization in ovaries but Krimp and Aub require each other for their proper nuage localization in testes. This suggests that the functional requirement of Krimp in piRNA biogenesis may be different in male and female gonads.
nuage; piRNA; PIWI; Drosophila; germline
Drosophila telomeres are maintained as a result of transpositions of specialized telomeric retrotransposons. The abundance of telomeric retroelement transcripts, as well as the frequency of their transpositions onto the chromosome ends, is controlled by a PIWI-interacting RNA (piRNA) pathway. In our recent report, we demonstrate strong evidence of piRNA-mediated transcriptional silencing of telomeric repeats in the Drosophila germline. Telomerase-generated repeats serve as a platform for recruiting specialized DNA-binding proteins which are involved in chromosome end protection and in the telomere length control. No specific proteins are known to bind to heterogeneous long sequences of the Drosophila telomeric retrotransposons. The importance of the piRNA silencing mechanism in the formation of telomeric chromatin along the region of the retrotransposon array will be discussed. We propose that Drosophila telomeric retrotransposon HeT-A serves as a template for the piRNA-mediated assembly of the specific protein complex, which is functionally similar to the recruiting of the DNA-binding telomeric proteins by the telomerase-generated repeats. The role of the piRNA pathway components in the assembly of the telomere capping complex was recently unveiled. Taken together, these data elucidate the importance of the piRNA pathway in the Drosophila telomere homeostasis.
Chromatin; Drosophila; germline; piRNA; PIWI; retrotransposon; telomere
The evolutionarily conserved Argonaute/PIWI (AGO/PIWI; a.k.a. PAZ-PIWI Domain, or PPD) family of proteins is crucial for the biogenesis and function of small non-coding RNAs (ncRNAs). This family can be divided into AGO and PIWI subfamilies. The AGO proteins are ubiquitously present in diverse tissues. They bind to small interfering RNAs (siRNAs) and microRNAs (miRNAs). In contrast, the PIWI proteins are predominantly present in the germline, and associate with a novel class of small RNAs known as PIWI-interacting RNAs (piRNAs). Tens of thousands of piRNA species, typically 24-32 nucleotide long, have been found in mammals, zebrafish, and Drosophila. Most piRNAs appear to be generated from a small number of long single-stranded RNA precursors that are often encoded by repetitive intergenic sequences in the genome. PIWI proteins play crucial roles during germline development and gametogenesis of many metazoan species, from germline determination and germline stem cell maintenance to meiosis, spermiogenesis, and transposon silencing. These diverse functions may involve piRNAs, and may be achieved via novel mechanisms of epigenetic and post-transcriptional regulation.
epigenetic regulation; germ cell; stem cell; transposon silencing; translational regulation
PIWI-interacting RNA (piRNA) clusters act as anti-transposon/retrovirus centers. Integration of selfish jumping elements into piRNA clusters generates de novo piRNAs, which in turn exert trans-silencing activity against these elements in animal gonads. To date, neither genome-wide chromatin modification states of piRNA clusters nor a mode for piRNA precursor transcription have been well understood. Here, to understand the chromatin landscape of piRNA clusters and how piRNA precursors are generated, we analyzed the transcriptome, transcription start sites (TSSs) and the chromatin landscape of the BmN4 cell line, which harbors the germ-line piRNA pathway. Notably, our epigenomic map demonstrated the highly euchromatic nature of unique piRNA clusters. RNA polymerase II was enriched for TSSs that transcribe piRNA precursors. piRNA precursors possessed 5′-cap structures as well as 3′-poly A-tails. Collectively, we envision that the euchromatic, opened nature of unique piRNA clusters or piRNA cluster-associated TSSs allows piRNA clusters to capture new insertions efficiently.
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.
Protecting the genome from transposable element (TE) mobilization is critical for germline development. In Drosophila, Piwi proteins and their bound small RNAs (piRNAs) provide a potent defense against TE activity. TE targeting piRNAs are processed from TE-dense heterochromatic loci termed ‘piRNA clusters’. While piRNA biogenesis from cluster precursors is beginning to be understood, little is known about piRNA cluster transcriptional regulation. Here we show that deposition of histone 3 lysine 9 by the methyltransferase dSETDB1 (egg) is required for piRNA cluster transcription. In the absence of dSETDB1, cluster precursor transcription collapses in germline and somatic gonadal cells and TEs are activated, resulting in germline loss and a block in germline stem cell differentiation. We propose that heterochromatin protects the germline by activating the piRNA pathway.
PIWI-interacting RNAs (piRNAs) provide defence against transposable element (TE) expansion in the germ line of metazoans. piRNAs are processed from the transcripts encoded by specialized heterochromatic clusters enriched in damaged copies of transposons. How these regions are recognized as a source of piRNAs is still elusive. The aim of this study is to determine how transgenes that contain a fragment of the Long Interspersed Nuclear Elements (LINE)-like I transposon lead to an acquired TE resistance in Drosophila. We show that such transgenes, being inserted in unique euchromatic regions that normally do not produce small RNAs, become de novo bidirectional piRNA clusters that silence I-element activity in the germ line. Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences—not only from the transposon fragment. Chromatin immunoprecipitation analysis shows that in ovaries, the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes producing piRNAs. We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs.
Piwi-interacting RNAs (piRNAs) are expressed in mammalian germline cells and have been identified as key players in germline development. These molecules, typically of length 25–33 nt, associate with Piwi proteins of the Argonaute family to form the Piwi-interacting RNA complex. These small regulatory RNAs have been implicated in spermatogenesis, repression of retrotransposon transposition in germline cells, epigenetic regulation and positive regulation of translation and mRNA stability. piRNABank is a highly user-friendly resource which stores empirically known sequences and other related information on piRNAs reported in human, mouse and rat. The database supports organism and chromosome-wise comprehensive search features including accession numbers, localization on chromosomes, gene name or symbol, sequence homology-based search, clusters and corresponding genes and repeat elements. It also displays each piRNA or piRNA cluster on a graphical genome-wide map (http://pirnabank.ibab.ac.in/).
Piwi-interacting RNAs (piRNAs) are known to regulate transposon activity in germ cells of several animal models that propagate sexually. However, the role of piRNAs during asexual reproduction remains almost unknown. Aphids that can alternate sexual and asexual reproduction cycles in response to seasonal changes of photoperiod provide a unique opportunity to study piRNAs and the piRNA pathway in both reproductive modes. Taking advantage of the recently sequenced genome of the pea aphid Acyrthosiphon pisum, we found an unusually large lineage-specific expansion of genes encoding the Piwi sub-clade of Argonaute proteins. In situ hybridisation showed differential expressions between the duplicated piwi copies: while Api-piwi2 and Api-piwi6 are “specialised” in germ cells their most closely related copy, respectively Api-piwi5 and Api-piwi3, are expressed in the somatic cells. The differential expression was also identified in duplicated ago3: Api-ago3a in germ cells and Api-ago3b in somatic cells. Moreover, analyses of expression profiles of the expanded piwi and ago3 genes by semi-quantitative RT-PCR showed that expressions varied according to the reproductive types. These specific expression patterns suggest that expanded aphid piwi and ago3 genes have distinct roles in asexual and sexual reproduction.
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.
piRNA; PIWI; LINE-1; Transposable element; Germline; Methylation; Epigenetics
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.
Epigenetic regulation by diverse classes of small RNAs is mediated by the highly conserved Argonaute/Piwi family of proteins. While Argonautes are broadly expressed, the Piwi subfamily primarily functions in the germ line. Piwi proteins are associated with germline-specific ribonucleoprotein (RNP) granules in Drosophila, zebrafish and mouse. Depending on the species and on the specific family member, Piwis play important roles in either spermatogenesis and/or in maintaining germ cell and stem cell totipotency. Piwis bind to a newly discovered class of small RNAs, called piRNAs. C. elegans contains a large set of Argonaute/Piwi related proteins, including two closely related to piwi, called prg-1 and prg-2. The function of prg-1 and prg-2, and whether piRNAs exist in C. elegans, is unknown.
Here, we demonstrate that the Piwi-like protein PRG-1 is localized to P granules in germ cells entering spermatogenesis, and is required for successful spermatogenesis. Loss of prg-1 causes a marked reduction in expression of a subset of mRNAs expressed during spermatogenesis, and prg-1 mutant sperm exhibit extensive defects in activation and fertilization. Moreover, prg-1 activity is required for the presence of the small RNAs called 21U-RNAs.
Our data suggest that PRG-1 promotes expression, processing, or stability of 21U-RNAs, which, in turn or in concert with PRG-1, promote proper expression of spermatogenesis transcripts.
C. elegans; germ line; piwi; 21U-RNA; spermatogenesis
Summary of Recent Advances
Argonaute is at the heart of all effector complexes in RNA interference. In the classical RNAi pathway Argonaute functions as the Slicer enzyme that cleaves a mRNA target directed by a complementary siRNA. Two recently described Argonaute protein subfamilies mediate distinct functions in RNAi. The Piwi subfamily functions in the germline through a novel class of small RNAs that are longer than Argonaute specific si- and miRNAs. Piwi-interacting RNAs (piRNAs) carry a 2’ O-methylation on their 3’ end and appear to be synthesized by a Piwi Slicer dependent mechanism. Piwi/piRNA complexes in mammals and flies are directly linked to the control of transposable elements during germline development. Amplified RNAi in C. elegans is mediated by secondary siRNAs selectively bound to secondary Argonautes (SAGOs) that belong to a worm specific Argonaute subfamily (WAGO). Secondary siRNAs are 5’ triphosphorylated which may allow specific loading into SAGO complexes that are rate limiting for RNAi in C. elegans. Interestingly SAGOs lack conserved Slicer amino acid residues and probably act in a Slicer-independent fashion.
Piwi proteins and Piwi-interacting small RNAs (piRNAs) have known functions in transposon silencing in the male germline of fetal and newborn mice. Both are also present in adult testes; however, their function here remains a mystery. Here, we confirm that most piRNAs in meiotic spermatocytes originate from clusters in non-repeat intergenic regions of DNA. The regulation of these piRNA clusters, including the processing of the precursor transcripts into individual piRNAs, is accomplished through mostly unknown processes. We present a possible regulatory mechanism for one such cluster, named cluster 1082B, located on chromosome 7 in the mouse genome. The 1082B precursor transcript and its 788 unique piRNAs are repressed by the Alkbh1 dioxygenase and the testis-specific transcription repressor Tzfp. We observe a remarkable >1000-fold upregulation of individual piRNAs in pachytene spermatocytes isolated from Alkbh1- and Tzfp-deficient murine testes. Repression of cluster 1082B is further supported by the identification of a 10-bp Tzfp recognition sequence contained within the precursor transcript. Downregulation of LINE1 and IAP transcripts in the Alkbh1- and Tzfp-deficient mice leads us to propose a potential role for the 1082B-encoded piRNAs in transposon control.