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1.  Drosophila Interspecific Hybrids Phenocopy piRNA-Pathway Mutants 
PLoS Biology  2012;10(11):e1001428.
Hybrids of two Drosophila species show transposable element derepression and piRNA pathway malfunction, revealing adaptive evolution of piRNA pathway components.
The Piwi-interacting RNA (piRNA) pathway defends the germline of animals from the deleterious activity of selfish transposable elements (TEs) through small-RNA mediated silencing. Adaptation to novel invasive TEs is proposed to occur by incorporating their sequences into the piRNA pool that females produce and deposit into their eggs, which then propagates immunity against specific TEs to future generations. In support of this model, the F1 offspring of crosses between strains of the same Drosophila species sometimes suffer from germline derepression of paternally inherited TE families, caused by a failure of the maternal strain to produce the piRNAs necessary for their regulation. However, many protein components of the Drosophila piRNA pathway exhibit signatures of positive selection, suggesting that they also contribute to the evolution of host genome defense. Here we investigate piRNA pathway function and TE regulation in the F1 hybrids of interspecific crosses between D. melanogaster and D. simulans and compare them with intraspecific control crosses of D. melanogaster. We confirm previous reports showing that intraspecific crosses are characterized by derepression of paternally inherited TE families that are rare or absent from the maternal genome and piRNA pool, consistent with the role of maternally deposited piRNAs in shaping TE silencing. In contrast to the intraspecific cross, we discover that interspecific hybrids are characterized by widespread derepression of both maternally and paternally inherited TE families. Furthermore, the pattern of derepression of TE families in interspecific hybrids cannot be attributed to their paucity or absence from the piRNA pool of the maternal species. Rather, we demonstrate that interspecific hybrids closely resemble piRNA effector-protein mutants in both TE misregulation and aberrant piRNA production. We suggest that TE derepression in interspecific hybrids largely reflects adaptive divergence of piRNA pathway genes rather than species-specific differences in TE-derived piRNAs.
Author Summary
Eukaryotic genomes contain large quantities of transposable elements (TEs), short self-replicating DNA sequences that can move within the genome. The selfish replication of TEs has potentially drastic consequences for the host, such as disruption of gene function, induction of sterility, and initiation or exacerbation of some cancers. Like the adaptive immune system that defends our bodies against pathogens, the Piwi-interacting RNA (piRNA) pathway defends animal genomes against the harmful effects of TEs. Fundamental to piRNA-mediated defense is the production of small noncoding RNAs that act like antibodies to target replicating TEs for destruction by piRNA-effector proteins. piRNAs are expected to diverge rapidly between species in response to genome infection by increasingly disparate TEs. Here, we tested this hypothesis by examining how differences in piRNAs between two species of fruit fly relate to TE “immunity” in their hybrid offspring. Because piRNAs are maternally deposited, we expected excessive replication of paternal TEs in hybrids. Surprisingly, we observe increased activity of both maternal and paternal TEs, together with defects in piRNA production that are reminiscent of piRNA effector-protein mutants. Our observations reveal that piRNA effector-proteins do not function properly in hybrids, and we propose that adaptive evolution among piRNA effector-proteins contributes to host genome defense and leads to the functional incompatibilities that we observe in hybrids.
doi:10.1371/journal.pbio.1001428
PMCID: PMC3506263  PMID: 23189033
2.  Diverse CRISPRs Evolving in Human Microbiomes 
PLoS Genetics  2012;8(6):e1002441.
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.
Author Summary
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.
doi:10.1371/journal.pgen.1002441
PMCID: PMC3374615  PMID: 22719260
3.  A Conserved Upstream Motif Orchestrates Autonomous, Germline-Enriched Expression of Caenorhabditis elegans piRNAs 
PLoS Genetics  2013;9(3):e1003392.
Piwi-interacting RNAs (piRNAs) fulfill a critical, conserved role in defending the genome against foreign genetic elements. In many organisms, piRNAs appear to be derived from processing of a long, polycistronic RNA precursor. Here, we establish that each Caenorhabditis elegans piRNA represents a tiny, autonomous transcriptional unit. Remarkably, the minimal C. elegans piRNA cassette requires only a 21 nucleotide (nt) piRNA sequence and an ∼50 nt upstream motif with limited genomic context for expression. Combining computational analyses with a novel, in vivo transgenic system, we demonstrate that this upstream motif is necessary for independent expression of a germline-enriched, Piwi-dependent piRNA. We further show that a single nucleotide position within this motif directs differential germline enrichment. Accordingly, over 70% of C. elegans piRNAs are selectively expressed in male or female germline, and comparison of the genes they target suggests that these two populations have evolved independently. Together, our results indicate that C. elegans piRNA upstream motifs act as independent promoters to specify which sequences are expressed as piRNAs, how abundantly they are expressed, and in what germline. As the genome encodes well over 15,000 unique piRNA sequences, our study reveals that the number of transcriptional units encoding piRNAs rivals the number of mRNA coding genes in the C. elegans genome.
Author Summary
Across the animal kingdom, Piwi-interacting small RNAs (piRNAs) protect genome integrity and promote fertility. While the functions of piRNAs are well-characterized, far less is known about how they are generated and how their expression is regulated. In the Caenorhabditis elegans genome, a conserved sequence motif lies upstream of many piRNA loci and appears to regulate their expression. We combined computational and experimental approaches to investigate the role of this motif in the expression of C. elegans piRNAs. We discovered that >70% of piRNAs are differentially enriched in male versus female germline, and these male and female piRNAs show different upstream motifs. Using a transgenic system for expressing synthetic piRNAs in vivo, we demonstrate that variation of a single nucleotide within this motif influences piRNA germline enrichment. We further show that the conserved motif is capable of driving piRNA expression in genomic isolation. Accordingly, the genomic distribution of these motifs determines which sequences are expressed as piRNAs in C. elegans. Our results suggest that each C. elegans piRNA represents an independent transcript whose sequence, abundance, and germline enrichment are encoded by a variant upstream motif, defining a novel modality for expression of piRNAs.
doi:10.1371/journal.pgen.1003392
PMCID: PMC3597512  PMID: 23516384
4.  A Complex of Cas Proteins 5, 6, and 7 Is Required for the Biogenesis and Stability of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-derived RNAs (crRNAs) in Haloferax volcanii* 
The Journal of Biological Chemistry  2014;289(10):7164-7177.
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.
doi:10.1074/jbc.M113.508184
PMCID: PMC3945376  PMID: 24459147
Archaea; Microbiology; Molecular Biology; Molecular Genetics; Protein Complexes; CRISPR/Cas; Cas6; Haloferax volcanii; crRNA; Type I-B
5.  Euchromatic Transposon Insertions Trigger Production of Novel Pi- and Endo-siRNAs at the Target Sites in the Drosophila Germline 
PLoS Genetics  2014;10(2):e1004138.
The control of transposable element (TE) activity in germ cells provides genome integrity over generations. A distinct small RNA–mediated pathway utilizing Piwi-interacting RNAs (piRNAs) suppresses TE expression in gonads of metazoans. In the fly, primary piRNAs derive from so-called piRNA clusters, which are enriched in damaged repeated sequences. These piRNAs launch a cycle of TE and piRNA cluster transcript cleavages resulting in the amplification of piRNA and TE silencing. Using genome-wide comparison of TE insertions and ovarian small RNA libraries from two Drosophila strains, we found that individual TEs inserted into euchromatic loci form novel dual-stranded piRNA clusters. Formation of the piRNA-generating loci by active individual TEs provides a more potent silencing response to the TE expansion. Like all piRNA clusters, individual TEs are also capable of triggering the production of endogenous small interfering (endo-si) RNAs. Small RNA production by individual TEs spreads into the flanking genomic regions including coding cellular genes. We show that formation of TE-associated small RNA clusters can down-regulate expression of nearby genes in ovaries. Integration of TEs into the 3′ untranslated region of actively transcribed genes induces piRNA production towards the 3′-end of transcripts, causing the appearance of genic piRNA clusters, a phenomenon that has been reported in different organisms. These data suggest a significant role of TE-associated small RNAs in the evolution of regulatory networks in the germline.
Author Summary
Silencing of transposable elements (TEs) in germ cells depends on a distinct class of small RNAs, Piwi-interacting RNAs (piRNAs). TE repression is provided by piRNAs derived from large heterochromatic loci enriched in fragmented TE copies, so-called piRNA clusters. According to the current model, individual TEs and their transcripts are considered merely as targets of cluster-derived primary piRNAs, which exert post-transcriptional and transcriptional silencing in Drosophila. In our work, we show that natural individual transposons become piRNA-generating loci themselves. We came to this conclusion by comparing the ovarian small RNAs and TE insertion sites of two Drosophila strains, which showed that euchromatic target sites of strain-specific TEs generate a number of novel strain-specific piRNAs. This mechanism allows production of additional small RNAs that target active TEs and provide more potent transposon suppression in the germline. Moreover, small RNA production by individual TEs spreads into the flanking genomic regions, which affects the expression of adjacent coding genes and microRNA genes. These data underline the role of individual TEs in a silencing response and explore a new level of TE impact on the gene regulatory networks in the germline.
doi:10.1371/journal.pgen.1004138
PMCID: PMC3916259  PMID: 24516406
6.  Cytoplasmic Compartmentalization of the Fetal piRNA Pathway in Mice 
PLoS Genetics  2009;5(12):e1000764.
Derepression of transposable elements (TEs) in the course of epigenetic reprogramming of the mouse embryonic germline necessitates the existence of a robust defense that is comprised of PIWI/piRNA pathway and de novo DNA methylation machinery. To gain further insight into biogenesis and function of piRNAs, we studied the intracellular localization of piRNA pathway components and used the combination of genetic, molecular, and cell biological approaches to examine the performance of the piRNA pathway in germ cells of mice lacking Maelstrom (MAEL), an evolutionarily conserved protein implicated in transposon silencing in fruit flies and mice. Here we show that principal components of the fetal piRNA pathway, MILI and MIWI2 proteins, localize to two distinct types of germinal cytoplasmic granules and exhibit differential association with components of the mRNA degradation/translational repression machinery. The first type of granules, pi-bodies, contains the MILI-TDRD1 module of the piRNA pathway and is likely equivalent to the enigmatic “cementing material” first described in electron micrographs of rat gonocytes over 35 years ago. The second type of granules, piP-bodies, harbors the MIWI2-TDRD9-MAEL module of the piRNA pathway and signature components of P-bodies, GW182, DCP1a, DDX6/p54, and XRN1 proteins. piP-bodies are found predominantly in the proximity of pi-bodies and the two frequently share mouse VASA homolog (MVH) protein, an RNA helicase. In Mael-mutant gonocytes, MIWI2, TDRD9, and MVH are lost from piP-bodies, whereas no effects on pi-body composition are observed. Further analysis revealed that MAEL appears to specifically facilitate MIWI2-dependent aspects of the piRNA pathway including biogenesis of secondary piRNAs, de novo DNA methylation, and efficient downregulation of TEs. Cumulatively, our data reveal elaborate cytoplasmic compartmentalization of the fetal piRNA pathway that relies on MAEL function.
Author Summary
Vast territories of animal genomes are populated by numerous types of mobile genetic elements (or transposons) that act predominantly as selfish parasites unconcerned with the impact of their activity on the well-being of the host. In response to the danger posed by transposons, organisms have evolved a defensive mechanism that employs a particular class of small RNAs known as piRNAs to identify and selectively silence transposons. We have studied the subcellular organization of such a defensive mechanism, the piRNA pathway, in germ cells of mouse male embryos. We discovered that key proteins involved in the genesis of small RNAs, MILI and MIWI2, occupy specific domains within the cytoplasm of germ cells. Surprisingly, MIWI2 shares its domain with proteins known to degrade RNAs and repress synthesis of cellular proteins, thus raising a possibility of cooperation of the two mechanisms in transposon defense. Genetic ablation of MAEL, a protein also found within the MIWI2 domain, disrupts normal MIWI2 localization and piRNA production leading to transposon activation. This study demonstrates that an elaborate compartmentalization of the defensive mechanism is required for the efficient recognition and destruction of active transposons in germ cells of mice.
doi:10.1371/journal.pgen.1000764
PMCID: PMC2785470  PMID: 20011505
7.  Type I-E CRISPR-Cas Systems Discriminate Target from Non-Target DNA through Base Pairing-Independent PAM Recognition 
PLoS Genetics  2013;9(9):e1003742.
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.
Author Summary
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.
doi:10.1371/journal.pgen.1003742
PMCID: PMC3764190  PMID: 24039596
8.  Substrate Generation for Endonucleases of CRISPR/Cas Systems 
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.
doi:10.3791/4277
PMCID: PMC3490271  PMID: 22986408
Molecular biology; Issue 67; CRISPR/Cas; endonuclease;  in vitro transcription; crRNA; Cas6
9.  CRISPR transcript processing: a mechanism for generating a large number of small interfering RNAs 
Biology Direct  2012;7:24.
Background
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.
Results
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.
Conclusions
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.
Reviewers
This article was reviewed by Mikhail Gelfand, Eugene Koonin and L Aravind.
doi:10.1186/1745-6150-7-24
PMCID: PMC3537551  PMID: 22849651
CRISPR/Cas; Transcript processing; Small RNA; CRISPR expression regulation; CRISPR/Cas response
10.  Programmable plasmid interference by the CRISPR-Cas system in Thermococcus kodakarensis 
RNA Biology  2013;10(5):828-840.
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.
doi:10.4161/rna.24084
PMCID: PMC3737340  PMID: 23535213
CRISPR; Cas; archaea; Thermococcus; hyperthermophile; immune; RNA; DNA; silencing; interference
11.  Cytotoxic Chromosomal Targeting by CRISPR/Cas Systems Can Reshape Bacterial Genomes and Expel or Remodel Pathogenicity Islands 
PLoS Genetics  2013;9(4):e1003454.
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.
Author Summary
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.
doi:10.1371/journal.pgen.1003454
PMCID: PMC3630108  PMID: 23637624
12.  Function and Regulation of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR Associated (Cas) Systems 
Viruses  2012;4(10):2291-2311.
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.
doi:10.3390/v4102291
PMCID: PMC3497052  PMID: 23202464
phages; plasmids; horizontal gene transfer; CRISPR; Cas; cascade; PAM; crRNA; resistance
13.  Computational Analysis of Mouse piRNA Sequence and Biogenesis 
PLoS Computational Biology  2007;3(11):e222.
The recent discovery of a new class of 30-nucleotide long RNAs in mammalian testes, called PIWI-interacting RNA (piRNA), with similarities to microRNAs and repeat-associated small interfering RNAs (rasiRNAs), has raised puzzling questions regarding their biogenesis and function. We report a comparative analysis of currently available piRNA sequence data from the pachytene stage of mouse spermatogenesis that sheds light on their sequence diversity and mechanism of biogenesis. We conclude that (i) there are at least four times as many piRNAs in mouse testes than currently known; (ii) piRNAs, which originate from long precursor transcripts, are generated by quasi-random enzymatic processing that is guided by a weak sequence signature at the piRNA 5′ends resulting in a large number of distinct sequences; and (iii) many of the piRNA clusters contain inverted repeats segments capable of forming double-strand RNA fold-back segments that may initiate piRNA processing analogous to transposon silencing.
Author Summary
The discovery of a new class of mammalian small regulatory RNAs termed PIWI-interacting RNA (piRNA) has extended the diverse family of small regulatory RNAs. PIWI proteins are a subclass of the larger Argonaute proteins family, of which the Ago members bind microRNAs and play a critical role in gene silencing. Despite the homology between PIWI and Ago proteins, piRNAs are strikingly different from microRNAs in their length, expression pattern, and genomic organization. In contrast, piRNAs are similar to repeat-associated small interfering RNA (rasiRNAs), a class of small RNAs that are responsible for transposon silencing in Drosophila germline, although it is unclear if piRNAs function in a similar way. This paper describes a computational comparison and analysis of the existing comprehensive piRNA datasets identified independently by three groups at the pachytene stage in mouse spermatogenesis. We find that the studies have identified similar genomic piRNA clusters, but differ substantially in the piRNAs that were cloned from those clusters. Based on these results we quantify the expected number of piRNAs and suggest that the processing of piRNAs from genomic transcripts is quasi-random. We find that a weak sequence signature may guide the piRNA 5′end processing that accounts for the departure from fully random processing. We further show partial evidence that piRNA biogenesis may be initiated by neighboring transposable elements.
doi:10.1371/journal.pcbi.0030222
PMCID: PMC2065894  PMID: 17997596
14.  Self vs. non-self discrimination during CRISPR RNA-directed immunity 
Nature  2010;463(7280):568-571.
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.
doi:10.1038/nature08703
PMCID: PMC2813891  PMID: 20072129
15.  Vreteno, a gonad-specific protein, is essential for germline development and primary piRNA biogenesis in Drosophila 
Development (Cambridge, England)  2011;138(18):4039-4050.
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.
doi:10.1242/dev.069187
PMCID: PMC3160098  PMID: 21831924
Germline stem cell; Soma; Transposon; Piwi; Aubergine; piRNAs; Tudor; Drosophila
16.  A broadly conserved pathway generates 3' UTR-directed primary piRNAs 
Current biology : CB  2009;19(24):2066-2076.
Summary
Background
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.
Results
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.
Conclusions
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.
doi:10.1016/j.cub.2009.11.064
PMCID: PMC2812478  PMID: 20022248
17.  Human piRNAs Are Under Selection in Africans and Repress Transposable Elements 
Molecular Biology and Evolution  2011;28(11):3061-3067.
Piwi-interacting RNAs (piRNAs) are a recently discovered class of 24- to 30-nt noncoding RNAs whose best-understood function is to repress transposable elements (TEs) in animal germ lines. In humans, TE-derived sequences comprise ∼45% of the genome and there are several active TE families, including LINE-1 and Alu elements, which are a significant source of de novo mutations and intrapopulation variability. In the “ping-pong model,” piRNAs are thought to alternatively cleave sense and antisense TE transcripts in a positive feedback loop. Because piRNAs are poorly conserved between closely related species, including human and chimpanzee, we took a population genomics approach to study piRNA function and evolution. We found strong statistical evidence that piRNA sequences are under selective constraint in African populations. We then mapped the piRNA sequences to human TE sequences and found strong correlations between the age of each LINE-1 and Alu subfamily and the number of piRNAs mapping to the subfamily. This result supports the idea that piRNAs function as repressors of TEs in humans. Finally, we observed a significant depletion of piRNA matches in the reverse transcriptase region of the consensus human LINE-1 element but not of the consensus mouse LINE-1 element. This result suggests that reverse transcriptase might have an endogenous role specific to humans. Overall, our results elucidate the function and evolution of piRNAs in humans and highlight the utility of population genomics analysis for studying this rapidly evolving genetic system.
doi:10.1093/molbev/msr141
PMCID: PMC3199439  PMID: 21613236
piRNAs; transposable elements; population genetics; selective constraint; Africans
18.  CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III 
Nature  2011;471(7340):602-607.
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.
doi:10.1038/nature09886
PMCID: PMC3070239  PMID: 21455174
19.  The Cutoff protein regulates piRNA cluster expression and piRNA production in the Drosophila germline 
The EMBO Journal  2011;30(22):4601-4615.
The Cutoff protein regulates piRNA cluster expression and piRNA production in the Drosophila germline
The identity and function of many factors involved in the piRNA pathway remain unknown. Here, in Drosophila, cutoff plays a role in regulating piRNA cluster transcript levels and biogenesis together with the heterochromatin protein Rhino.
In a broad range of organisms, Piwi-interacting RNAs (piRNAs) have emerged as core components of a surveillance system that protects the genome by silencing transposable and repetitive elements. A vast proportion of piRNAs is produced from discrete genomic loci, termed piRNA clusters, which are generally embedded in heterochromatic regions. The molecular mechanisms and the factors that govern their expression are largely unknown. Here, we show that Cutoff (Cuff), a Drosophila protein related to the yeast transcription termination factor Rai1, is essential for piRNA production in germline tissues. Cuff accumulates at centromeric/pericentromeric positions in germ-cell nuclei and strongly colocalizes with the major heterochromatic domains. Remarkably, we show that Cuff is enriched at the dual-strand piRNA cluster 1/42AB and is likely to be involved in regulation of transcript levels of similar loci dispersed in the genome. Consistent with this observation, Cuff physically interacts with the Heterochromatin Protein 1 (HP1) variant Rhino (Rhi). Our results unveil a link between Cuff activity, heterochromatin assembly and piRNA cluster expression, which is critical for stem-cell and germ-cell development in Drosophila.
doi:10.1038/emboj.2011.334
PMCID: PMC3243597  PMID: 21952049
cutoff; Drosophila; germline; heterochromatin; piRNA
20.  RcsB-BglJ-mediated activation of Cascade operon does not induce the maturation of CRISPR RNAs in E. coli K12 
RNA Biology  2013;10(5):708-715.
Prokaryotic immunity against foreign nucleic acids mediated by clustered, regularly interspaced, short palindromic repeats (CRISPR) depends on the expression of the CRISPR-associated (Cas) proteins and the formation of small CRISPR RNAs (crRNAs). The crRNA-loaded Cas ribonucleoprotein complexes convey the specific recognition and inactivation of target nucleic acids. In E. coli K12, the maturation of crRNAs and the interference with target DNA is performed by the Cascade complex. The transcription of the Cascade operon is tightly repressed through H-NS-dependent inhibition of the Pcas promoter. Elevated levels of the LysR-type regulator LeuO induce the Pcas promoter and concomitantly activate the CRISPR-mediated immunity against phages. Here, we show that the Pcas promoter can also be induced by constitutive expression of the regulator BglJ. This activation is LeuO-dependent as heterodimers of BglJ and RcsB activate leuO transcription. Each transcription factor, LeuO or BglJ, induced the transcription of the Cascade genes to comparable amounts. However, the maturation of the crRNAs was activated in LeuO but not in BglJ-expressing cells. Studies on CRISPR promoter activities, transcript stabilities, crRNA processing and Cascade protein levels were performed to answer the question why crRNA maturation is defective in BglJ-expressing cells. Our results demonstrate that the activation of Cascade gene transcription is necessary but not sufficient to turn on the CRISPR-mediated immunity and suggest a more complex regulation of the type I-E CRISPR-Cas system in E. coli.
doi:10.4161/rna.23765
PMCID: PMC3737329  PMID: 23392250
CRISPR; Cas protein; Cascade; H-NS; LeuO; transcription regulation
21.  Rapid Evolution of piRNA Pathway in the Teleost Fish: Implication for an Adaptation to Transposon Diversity 
Genome Biology and Evolution  2014;6(6):1393-1407.
The Piwi-interacting RNA (piRNA) pathway is responsible for germline specification, gametogenesis, transposon silencing, and genome integrity. Transposable elements can disrupt genome and its functions. However, piRNA pathway evolution and its adaptation to transposon diversity in the teleost fish remain unknown. This article unveils evolutionary scene of piRNA pathway and its association with diverse transposons by systematically comparative analysis on diverse teleost fish genomes. Selective pressure analysis on piRNA pathway and miRNA/siRNA (microRNA/small interfering RNA) pathway genes between teleosts and mammals showed an accelerated evolution of piRNA pathway genes in the teleost lineages, and positive selection on functional PAZ (Piwi/Ago/Zwille) and Tudor domains involved in the Piwi–piRNA/Tudor interaction, suggesting that the amino acid substitutions are adaptive to their functions in piRNA pathway in the teleost fish species. Notably five piRNA pathway genes evolved faster in the swamp eel, a kind of protogynous hermaphrodite fish, than the other teleosts, indicating a differential evolution of piRNA pathway between the swamp eel and other gonochoristic fishes. In addition, genome-wide analysis showed higher diversity of transposons in the teleost fish species compared with mammals. Our results suggest that rapidly evolved piRNA pathway in the teleost fish is likely to be involved in the adaption to transposon diversity.
doi:10.1093/gbe/evu105
PMCID: PMC4079211  PMID: 24846630
teleost fish; evolution; positive selection; reproduction
22.  HP1 Recruitment in the Absence of Argonaute Proteins in Drosophila 
PLoS Genetics  2010;6(3):e1000880.
Highly repetitive and transposable element rich regions of the genome must be stabilized by the presence of heterochromatin. A direct role for RNA interference in the establishment of heterochromatin has been demonstrated in fission yeast. In metazoans, which possess multiple RNA–silencing pathways that are both functionally distinct and spatially restricted, whether RNA silencing contributes directly to heterochromatin formation is not clear. Previous studies in Drosophila melanogaster have suggested the involvement of both the AGO2-dependent endogenous small interfering RNA (endo-siRNA) as well as Piwi-interacting RNA (piRNA) silencing pathways. In order to determine if these Argonaute genes are required for heterochromatin formation, we utilized transcriptional reporters and chromatin immunoprecipitation of the critical factor Heterochromatin Protein 1 (HP1) to monitor the heterochromatic state of piRNA clusters, which generate both endo-siRNAs and the bulk of piRNAs. Surprisingly, we find that mutation of AGO2 or piwi increases silencing at piRNA clusters corresponding to an increase of HP1 association. Furthermore, loss of piRNA production from a single piRNA cluster results in genome-wide redistribution of HP1 and reduction of silencing at a distant heterochromatic site, suggesting indirect effects on HP1 recruitment. Taken together, these results indicate that heterochromatin forms independently of endo-siRNA and piRNA pathways.
Author Summary
One role for silent heterochromatin is to preserve the integrity of the genome by stabilizing regions rich in repetitive sequence and mobile elements. Compaction of repetitive sequences by heterochromatin is needed to prevent genome rearrangement and loss of genetic material. Furthermore, uncontrolled movement of mobile elements throughout the genome can result in deleterious mutations. In fission yeast, one important mechanism of heterochromatin establishment occurs through RNA interference, an RNA–dependent gene silencing process. However, it is unclear whether a direct role for RNA silencing in heterochromatin formation is conserved throughout evolution. In the fruit fly, Drosophila melanogaster, which harbors multiple RNA–silencing pathways that are both functionally distinct and spatially restricted, previous studies have suggested the involvement of the endogenous small interfering RNA (endo-siRNA) and Piwi-interacting RNA (piRNA) pathways in heterochromatin formation. These small RNA silencing pathways suppress the expression of mobile elements in the soma or in both somatic and germline tissues, respectively. Utilizing complementary genetic and biochemical approaches, we monitored the heterochromatin state at discrete genomic locations from which both types of these small RNAs originate in endo-siRNA or piRNA pathway mutants. Our results indicate that heterochromatin can form independently of these two small RNA silencing pathways.
doi:10.1371/journal.pgen.1000880
PMCID: PMC2837403  PMID: 20300658
23.  C. elegans piRNAs mediate the genome-wide surveillance of germline transcripts 
Cell  2012;150(1):78-87.
SUMMARY
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.
doi:10.1016/j.cell.2012.06.016
PMCID: PMC3410639  PMID: 22738724
24.  Comparative analysis of Cas6b processing and CRISPR RNA stability 
RNA Biology  2013;10(5):700-707.
The prokaryotic antiviral defense systems CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) employs short crRNAs (CRISPR RNAs) to target invading viral nucleic acids. A short spacer sequence of these crRNAs can be derived from a viral genome and recognizes a reoccurring attack of a virus via base complementarity. We analyzed the effect of spacer sequences on the maturation of crRNAs of the subtype I-B Methanococcus maripaludis C5 CRISPR cluster. The responsible endonuclease, termed Cas6b, bound non-hydrolyzable repeat RNA as a dimer and mature crRNA as a monomer. Comparative analysis of Cas6b processing of individual spacer-repeat-spacer RNA substrates and crRNA stability revealed the potential influence of spacer sequence and length on these parameters. Correlation of these observations with the variable abundance of crRNAs visualized by deep-sequencing analyses is discussed. Finally, insertion of spacer and repeat sequences with archaeal poly-T termination signals is suggested to be prevented in archaeal CRISPR/Cas systems.
doi:10.4161/rna.23715
PMCID: PMC3737328  PMID: 23392318
CRISPR; Cas6; endonuclease; crRNA; in-line probing; RNA binding; transcription termination
25.  piRNAs and epigenetic conversion in Drosophila 
Fly  2013;7(4):237-241.
Transposable element (TE) activity is repressed in the Drosophila germline by Piwi-Interacting RNAs (piRNAs), a class of small non-coding RNAs. These piRNAs are produced by discrete genomic loci containing TE fragments. In a recent publication, we tested for the existence of a strict epigenetic induction of piRNA production capacity by a locus in the D. melanogaster genome. We used 2 lines carrying a transgenic 7-copy tandem cluster (P-lacZ-white) at the same genomic site. This cluster generates in both lines a local heterochromatic sector. One line (T-1) produces high levels of ovarian piRNAs homologous to the P-lacZ-white transgenes and shows a strong capacity to repress homologous sequences in trans, whereas the other line (BX2) is devoid of both of these capacities. The properties of these 2 lines are perfectly stable over generations. We have shown that the maternal transmission of a cytoplasm carrying piRNAs from the first line can confer to the inert transgenic locus of the second, a totally de novo capacity to produce high levels of piRNAs as well as the ability to induce homology-dependent silencing in trans. These new properties are stably inherited over generations (n > 50). Furthermore, the converted locus has itself become able to convert an inert transgenic locus via cytoplasmic maternal inheritance. This results in a stable epigenetic conversion process, which can be performed recurrently—a phenomenon termed paramutation and discovered in Maize 60 y ago. Paramutation in Drosophila corresponds to the first stable paramutation in animals and provides a model system to investigate the epigenetically induced emergence of a piRNA-producing locus, a crucial step in epigenome shaping. In this Extra View, we discuss some additional functional aspects and the possible molecular mechanism of this piRNA-linked paramutation.
doi:10.4161/fly.26522
PMCID: PMC3896495  PMID: 24088599
epigenetics; cellular memory; heterochromatin; piRNAs; transposable elements

Results 1-25 (817661)