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.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, together with cas (CRISPR–associated) genes, form the CRISPR/Cas adaptive immune system, a primary defense strategy that eubacteria and archaea mobilize against foreign nucleic acids, including phages and conjugative plasmids. Short spacer sequences separated by the repeats are derived from foreign DNA and direct interference to future infections. The availability of hundreds of shotgun metagenomic datasets from the Human Microbiome Project (HMP) enables us to explore the distribution and diversity of known CRISPRs in human-associated microbial communities and to discover new CRISPRs. We propose a targeted assembly strategy to reconstruct CRISPR arrays, which whole-metagenome assemblies fail to identify. For each known CRISPR type (identified from reference genomes), we use its direct repeat consensus sequence to recruit reads from each HMP dataset and then assemble the recruited reads into CRISPR loci; the unique spacer sequences can then be extracted for analysis. We also identified novel CRISPRs or new CRISPR variants in contigs from whole-metagenome assemblies and used targeted assembly to more comprehensively identify these CRISPRs across samples. We observed that the distributions of CRISPRs (including 64 known and 86 novel ones) are largely body-site specific. We provide detailed analysis of several CRISPR loci, including novel CRISPRs. For example, known streptococcal CRISPRs were identified in most oral microbiomes, totaling ∼8,000 unique spacers: samples resampled from the same individual and oral site shared the most spacers; different oral sites from the same individual shared significantly fewer, while different individuals had almost no common spacers, indicating the impact of subtle niche differences on the evolution of CRISPR defenses. We further demonstrate potential applications of CRISPRs to the tracing of rare species and the virus exposure of individuals. This work indicates the importance of effective identification and characterization of CRISPR loci to the study of the dynamic ecology of microbiomes.
Human bodies are complex ecological systems in which various microbial organisms and viruses interact with each other and with the human host. The Human Microbiome Project (HMP) has resulted in >700 datasets of shotgun metagenomic sequences, from which we can learn about the compositions and functions of human-associated microbial communities. CRISPR/Cas systems are a widespread class of adaptive immune systems in bacteria and archaea, providing acquired immunity against foreign nucleic acids: CRISPR/Cas defense pathways involve integration of viral- or plasmid-derived DNA segments into CRISPR arrays (forming spacers between repeated structural sequences), and expression of short crRNAs from these single repeat-spacer units, to generate interference to future invading foreign genomes. Powered by an effective computational approach (the targeted assembly approach for CRISPR), our analysis of CRISPR arrays in the HMP datasets provides the very first global view of bacterial immunity systems in human-associated microbial communities. The great diversity of CRISPR spacers we observed among different body sites, in different individuals, and in single individuals over time, indicates the impact of subtle niche differences on the evolution of CRISPR defenses and indicates the key role of bacteriophage (and plasmids) in shaping human microbial communities.
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.
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.
RNA interference-related silencing mechanisms concern very diverse and distinct biological processes, from gene regulation (via the microRNA pathway) to defense against molecular parasites (through the small interfering RNA and the Piwi-interacting RNA pathways). Small non-coding RNAs serve as specificity factors that guide effector proteins to ribonucleic acid targets via base-pairing interactions, to achieve transcriptional or post-transcriptional regulation. Because of the small sequence complementarity required for microRNA-dependent post-transcriptional regulation, thousands of microRNA (miRNA) putative targets have been annotated in Drosophila. In Drosophila somatic ovarian cells, genomic parasites, such as transposable elements (TEs), are transcriptionally repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs) that prevent them from invading the germinal genome. Here we show, for the first time, that a functional miRNA pathway is required for the piRNA-mediated transcriptional silencing of TEs in this tissue. Global miRNA depletion, caused by tissue- and stage-specific knock down of drosha (involved in miRNA biogenesis), AGO1 or gawky (both responsible for miRNA activity), resulted in loss of TE-derived piRNAs and chromatin-mediated transcriptional de-silencing of TEs. This specific TE de-repression was also observed upon individual titration (by expression of the complementary miRNA sponge) of two miRNAs (miR-14 and miR-34) as well as in a miR-14 loss-of-function mutant background. Interestingly, the miRNA defects differentially affected TE- and 3' UTR-derived piRNAs. To our knowledge, this is the first indication of possible differences in the biogenesis or stability of TE- and 3' UTR-derived piRNAs. This work is one of the examples of detectable phenotypes caused by loss of individual miRNAs in Drosophila and the first genetic evidence that miRNAs have a role in the maintenance of genome stability via piRNA-mediated TE repression.
The fine-tuning of gene expression required for the normal development of multicellular organisms involves small RNAs that are called microRNAs (miRNAs). MiRNAs can reduce the stability or the activity of the many cellular messenger RNAs that contain miRNA complementary sequences. In animal gonads, the harmful expression and proliferation of genomic parasites, such as transposable elements, is prevented by a similar, sequence homology-based silencing mechanism that involves a different class of small RNAs, the Piwi-interacting RNAs (piRNAs). We report here that, in Drosophila somatic ovarian tissues, two miRNAs, miR-14 and miR-34, are required for the accumulation of piRNAs that prevent the expression of transposable elements and, probably, the subsequent invasion of the germinal genome. On the other hand, we found that other sources of piRNA production, such as the 3' end of genes, are miRNA-independent, suggesting the existence of variations in the piRNA biogenesis pathways depending on the piRNA genomic origin. Our results therefore highlight a novel miRNA function in the maintenance of genome stability through piRNA-mediated TE repression.
Background: The Cas6 protein is required for generating crRNAs in CRISPR-Cas I and III systems.
Results: The Cas6 protein is necessary for crRNA production but not sufficient for crRNA maintenance in Haloferax.
Conclusion: A Cascade-like complex is required in the type I-B system for a stable crRNA population.
Significance: The CRISPR-Cas system I-B has a similar Cascade complex like types I-A and I-E.
The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1–8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA.
Archaea; Microbiology; Molecular Biology; Molecular Genetics; Protein Complexes; CRISPR/Cas; Cas6; Haloferax volcanii; crRNA; Type I-B
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.
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.
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.
piRNA; transposable elements; Drosophila melanogaster; de novo piRNA production
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.
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.
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.
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.
The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1)1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.
Molecular biology; Issue 67; CRISPR/Cas; endonuclease; in vitro transcription; crRNA; Cas6
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.
Germline; Nuage; piRNA; Tudor domain
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.
transposable elements; RNAi; genome evolution
Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from Staphylococcus epidermidis has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from Escherichia coli we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism.
CRISPR loci and their associated genes form a diverse set of adaptive immune systems that are widespread among prokaryotes. In these systems, the CRISPR-associated genes (cas) encode for proteins that capture fragments of invading DNA and integrate these sequences between repeat sequences of the host's CRISPR locus. This information is used upon re-infection to degrade invader genomes. Storing invader sequences in host genomes necessitates a mechanism to differentiate between invader sequences on invader genomes and invader sequences on the host genome. CRISPR-Cas of Staphylococcus epidermidis (Type III-A system) is inhibited when invader sequences are flanked by repeat sequences, and this prevents targeting of the CRISPR locus on the host genome. Here we demonstrate that Escherichia coli CRISPR-Cas (Type I-E system) is not inhibited by repeat sequences. Instead, this system is specifically activated by the presence of bona fide Protospacer Adjacent Motifs (PAMs) in the target. PAMs are conserved sequences adjoining invader sequences on the invader genome, and these sequences are never adjacent to invader sequences within host CRISPR loci. PAM recognition is not affected by base pairing potential of the target with the crRNA. As such, the Type I-E system lacks the ability to specifically recognize self DNA.
CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated sequences) is a recently discovered prokaryotic defense system against foreign DNA, including viruses and plasmids. CRISPR cassette is transcribed as a continuous transcript (pre-crRNA), which is processed by Cas proteins into small RNA molecules (crRNAs) that are responsible for defense against invading viruses. Experiments in E. coli report that overexpression of cas genes generates a large number of crRNAs, from only few pre-crRNAs.
We here develop a minimal model of CRISPR processing, which we parameterize based on available experimental data. From the model, we show that the system can generate a large amount of crRNAs, based on only a small decrease in the amount of pre-crRNAs. The relationship between the decrease of pre-crRNAs and the increase of crRNAs corresponds to strong linear amplification. Interestingly, this strong amplification crucially depends on fast non-specific degradation of pre-crRNA by an unidentified nuclease. We show that overexpression of cas genes above a certain level does not result in further increase of crRNA, but that this saturation can be relieved if the rate of CRISPR transcription is increased. We furthermore show that a small increase of CRISPR transcription rate can substantially decrease the extent of cas gene activation necessary to achieve a desired amount of crRNA.
The simple mathematical model developed here is able to explain existing experimental observations on CRISPR transcript processing in Escherichia coli. The model shows that a competition between specific pre-crRNA processing and non-specific degradation determines the steady-state levels of crRNA and is responsible for strong linear amplification of crRNAs when cas genes are overexpressed. The model further shows how disappearance of only a few pre-crRNA molecules normally present in the cell can lead to a large (two orders of magnitude) increase of crRNAs upon cas overexpression. A crucial ingredient of this large increase is fast non-specific degradation by an unspecified nuclease, which suggests that a yet unidentified nuclease(s) is a major control element of CRISPR response. Transcriptional regulation may be another important control mechanism, as it can either increase the amount of generated pre-crRNA, or alter the level of cas gene activity.
This article was reviewed by Mikhail Gelfand, Eugene Koonin and L Aravind.
CRISPR/Cas; Transcript processing; Small RNA; CRISPR expression regulation; CRISPR/Cas response
CRISPR-Cas systems are RNA-guided immune systems that protect prokaryotes against viruses and other invaders. The CRISPR locus encodes crRNAs that recognize invading nucleic acid sequences and trigger silencing by the associated Cas proteins. There are multiple CRISPR-Cas systems with distinct compositions and mechanistic processes. Thermococcus kodakarensis (Tko) is a hyperthermophilic euryarchaeon that has both a Type I-A Csa and a Type I-B Cst CRISPR-Cas system. We have analyzed the expression and composition of crRNAs from the three CRISPRs in Tko by RNA deep sequencing and northern analysis. Our results indicate that crRNAs associated with these two CRISPR-Cas systems include an 8-nucleotide conserved sequence tag at the 5′ end. We challenged Tko with plasmid invaders containing sequences targeted by endogenous crRNAs and observed active CRISPR-Cas-mediated silencing. Plasmid silencing was dependent on complementarity with a crRNA as well as on a sequence element found immediately adjacent to the crRNA recognition site in the target termed the PAM (protospacer adjacent motif). Silencing occurred independently of the orientation of the target sequence in the plasmid, and appears to occur at the DNA level, presumably via DNA degradation. In addition, we have directed silencing of an invader plasmid by genetically engineering the chromosomal CRISPR locus to express customized crRNAs directed against the plasmid. Our results support CRISPR engineering as a feasible approach to develop prokaryotic strains that are resistant to infection for use in industry.
CRISPR; Cas; archaea; Thermococcus; hyperthermophile; immune; RNA; DNA; silencing; interference
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.
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.
In Drosophila, Piwi proteins associate with Piwi-interacting RNAs (piRNAs) and protect the germline genome by silencing mobile genetic elements. This defense system acts in germline and gonadal somatic tissue to preserve germline development. Genetic control for these silencing pathways varies greatly between tissues of the gonad. Here, we identified Vreteno (Vret), a novel gonad-specific protein essential for germline development. Vret is required for piRNA-based transposon regulation in both germline and somatic gonadal tissues. We show that Vret, which contains Tudor domains, associates physically with Piwi and Aubergine (Aub), stabilizing these proteins via a gonad-specific mechanism that is absent in other fly tissues. In the absence of vret, Piwi-bound piRNAs are lost without changes in piRNA precursor transcript production, supporting a role for Vret in primary piRNA biogenesis. In the germline, piRNAs can engage in an Aub- and Argonaute 3 (AGO3)-dependent amplification in the absence of Vret, suggesting that Vret function can distinguish between primary piRNAs loaded into Piwi-Aub complexes and piRNAs engaged in the amplification cycle. We propose that Vret plays an essential role in transposon regulation at an early stage of primary piRNA processing.
Germline stem cell; Soma; Transposon; Piwi; Aubergine; piRNAs; Tudor; Drosophila
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.
Phages are the most abundant biological entities on earth and pose a constant challenge to their bacterial hosts. Thus, bacteria have evolved numerous ‘innate’ mechanisms of defense against phage, such as abortive infection or restriction/modification systems. In contrast, the clustered regularly interspaced short palindromic repeats (CRISPR) systems provide acquired, yet heritable, sequence-specific ‘adaptive’ immunity against phage and other horizontally-acquired elements, such as plasmids. Resistance is acquired following viral infection or plasmid uptake when a short sequence of the foreign genome is added to the CRISPR array. CRISPRs are then transcribed and processed, generally by CRISPR associated (Cas) proteins, into short interfering RNAs (crRNAs), which form part of a ribonucleoprotein complex. This complex guides the crRNA to the complementary invading nucleic acid and targets this for degradation. Recently, there have been rapid advances in our understanding of CRISPR/Cas systems. In this review, we will present the current model(s) of the molecular events involved in both the acquisition of immunity and interference stages and will also address recent progress in our knowledge of the regulation of CRISPR/Cas systems.
phages; plasmids; horizontal gene transfer; CRISPR; Cas; cascade; PAM; crRNA; resistance
All immune systems must distinguish self from non-self to repel invaders without inducing autoimmunity. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci protect bacteria and archaea from invasion by phage and plasmid DNA through a genetic interference pathway1–9. CRISPR loci are present in ~ 40% and ~90% of sequenced bacterial and archaeal genomes respectively10 and evolve rapidly, acquiring new spacer sequences to adapt to highly dynamic viral populations1, 11–13. Immunity requires a sequence match between the invasive DNA and the spacers that lie between CRISPR repeats1–9. Each cluster is genetically linked to a subset of the cas (CRISPR-associated) genes14–16 that collectively encode >40 families of proteins involved in adaptation and interference. CRISPR loci encode small CRISPR RNAs (crRNAs) that contain a full spacer flanked by partial repeat sequences2, 17–19. CrRNA spacers are thought to identify targets by direct Watson-Crick pairing with invasive “protospacer” DNA2, 3, but how they avoid targeting the spacer DNA within the encoding CRISPR locus itself is unknown. Here we have defined the mechanism of CRISPR self/non-self discrimination. In Staphylococcus epidermidis, target/crRNA mismatches at specific positions outside of the spacer sequence license foreign DNA for interference, whereas extended pairing between crRNA and CRISPR DNA repeats prevents autoimmunity. Hence, this CRISPR system uses the base-pairing potential of crRNAs not only to specify a target but also to spare the bacterial chromosome from interference. Differential complementarity outside of the spacer sequence is a built-in feature of all CRISPR systems, suggesting that this mechanism is a broadly applicable solution to the self/non-self dilemma that confronts all immune pathways.
In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas–mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA–targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity.
Bacteria have evolved mechanisms that provide protection from continual invasion by viruses and other foreign elements. Resistance systems, known as CRISPR/Cas, were recently discovered and equip bacteria and archaea with an “adaptive immune system.” This adaptive immunity provides a highly evolvable sequence-specific small RNA–based memory of past invasions by viruses and foreign genetic elements. There are many cases where these systems appear to target regions within the bacterial host's own genome (a possible autoimmunity), but the evolutionary rationale for this is unclear. Here, we demonstrate that CRISPR/Cas targeting of the host chromosome is highly toxic but that cells survive through mutations that alleviate the immune mechanism. We have used this phenotype to gain insight into how these systems function and show that large changes in the bacterial genome can occur. For example, targeting of a chromosomal pathogenicity island, important for virulence of the potato pathogen Pectobacterium atrosepticum, resulted in deletion of the island, which constituted ∼2% of the bacterial genome. These results have broad significance for the role of CRISPR/Cas systems and their impact on the evolution of bacterial genomes and virulence. In addition, this study demonstrates their potential as a tool for the targeted deletion of specific regions of bacterial chromosomes.
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
CRISPR/Cas systems constitute a widespread class of immunity systems that protect bacteria and archaea against phages and plasmids, and commonly use repeat/spacer-derived short crRNAs to silence foreign nucleic acids in a sequence-specific manner. Although the maturation of crRNAs represents a key event in CRISPR activation, the responsible endoribonucleases (CasE, Cas6, Csy4) are missing in many CRISPR/Cas subtypes. Here, differential RNA sequencing of the human pathogen Streptococcus pyogenes uncovered tracrRNA, a trans-encoded small RNA with 24 nucleotide complementarity to the repeat regions of crRNA precursor transcripts. We show that tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein; all these components are essential to protect S. pyogenes against prophage-derived DNA. Our study reveals a novel pathway of small guide RNA maturation and the first example of a host factor (RNase III) required for bacterial RNA-mediated immunity against invaders.
Piwi-interacting (pi-) RNAs guide germline-expressed Piwi proteins in order to suppress the activity of transposable elements (TEs). But notably, the majority of pachytene piRNAs in mammalian testes is not related to TEs. This raises the question of whether the Piwi/piRNA pathway exerts functions beyond TE silencing. Although gene-derived piRNAs were described many times, a possible gene-regulatory function was doubted due to the absence of antisense piRNAs. Here we sequenced and analyzed piRNAs expressed in the adult testis of the pig, as this taxon possesses the full set of mammalian Piwi paralogs while their spermatozoa are marked by an extreme fitness due to selective breeding. We provide an exhaustive characterization of porcine piRNAs and genomic piRNA clusters. Moreover, we reveal that both sense and antisense piRNAs derive from protein-coding genes, while exhibiting features that clearly show that they originate from the Piwi/piRNA-mediated post-transcriptional silencing pathway, commonly referred to as ping-pong cycle. We further show that the majority of identified piRNA clusters in the porcine genome spans exonic sequences of protein-coding genes or pseudogenes, which reveals a mechanism by which primary antisense piRNAs directed against mRNA can be generated. Our data provide evidence that spliced mRNAs, derived from such loci, are not only targeted by piRNAs but are also subject to ping-pong cycle processing. Finally, we demonstrate that homologous genes are targeted and processed by piRNAs in pig, mouse and human. Altogether, this strongly suggests a conserved role for the mammalian Piwi/piRNA pathway in post-transcriptional regulation of protein-coding genes, which did not receive much attention so far.