Although hints of the piRNA system were observed in Drosophila
as early as 2001 [13
], piRNAs were definitively discovered by several groups independently in 2006 by immunoprecipitating Piwi protein from mammalian testis and sequencing the bound small RNAs [14–17
]. PiRNAs are ~26–30
nt in mammals although their lengths can be slightly different in other animals. PiRNAs have essentially no known defining sequence characteristics beyond a very strong propensity for a 5′-uridine and a weaker bias toward an adenosine at position 10. PiRNAs are in general difficult to predict bioinformatically and must instead be defined biochemically. However, protocols for immunoprecipitating Piwi protein are still an active area of research [18
] and there are no definitive sets of piRNA genes yet because the population of piRNAs is typically very large (in the hundreds of thousands) and complex. Caenorhabditis
piRNAs may be significantly different from mammalian and Drosophila
piRNAs because they have a different length (21
nt), and there appears to be a conserved promoter motif upstream of many piRNAs [19
], suggesting that each piRNA is a separate transcription unit, unlike piRNAs in mammals and Drosophila
which are typically expressed in long polycistronic transcripts.
Unlike other small RNAs from the RNAi-related pathway, such as microRNAs and small interfering RNAs, which are produced from double-stranded intermediates by the Dicer enzyme, piRNAs are thought to be produced from long polycistronic RNA transcripts by a Dicer-independent mechanism in mammals and Drosophila
. Note that unlike the CRISPR system described below, piRNA populations are very complex and piRNAs appear to be produced by quasi-random cleavage of the primary piRNA transcript [20
]. That is, while piRNAs almost always start with a U and there are biases for particular sequences to be cleaved as piRNAs, there is a strong random component that determines which sequences of the primary transcript are processed into piRNAs (hence the term ‘quasi-random’). PiRNA 3′-end formation is poorly understood and is an object of active research [21
]. However, piRNA 5′-end formation was addressed by several key papers [22
]. The authors studied master loci that control transposable element proliferation in Drosophila
but were molecularly uncharacterized for many years because of the apparent lack of functional sequences at the loci, other than a jumble of transposable element insertions. These master loci were found to produce piRNAs that repress transposable elements in trans
] (). The authors proposed the Ping-Pong mechanism [22
] in which primary piRNAs cleave sense transposon transcripts and simultaneously produce secondary piRNAs from the sense transposons that then cleave antisense transposon transcripts. This mechanism thus depends on the transcription of both sense and anti-sense transposon transcripts. An alternate view is that piRNAs are in fact produced through a double-stranded intermediate [24
] based on the recent reports of the existence of an RNA-dependent RNA polymerase in Drosophila
]. However, the existence of a Drosophila
RDRP remains controversial, and this view remains a minority interpretation at the present time.
Figure 1: PiRNAs expressed from discrete loci in the Drosophila genome (X-TAS and Flamenco) repress transposable elements in trans (gypsy, P-element, Idefix, ZAM). Reprinted from ‘Mighty Piwis Defend Germline against Genome Intruders’, K. A. O’Donnell (more ...)
Since the Ping-Pong mechanism is a positive feedback loop, one question is how the Ping-Pong mechanism is started in the first place. In Drosophila
, a partial answer is provided by the fact that piRNAs are deposited maternally into the embryo [26
]. PiRNAs can thus be inherited epigenetically across generations. A second answer comes from evidence in Drosophila
, where primary piRNAs are produced in the somatic follicle cells and delivered to the germline to start the Ping-Pong cycle [28
]. A similar mechanism is found in Arabidopsis
for a different class of small RNAs [30
], suggesting that this may be a universal mechanism where transposons are activated outside of the germline to generate small RNAs, thus reducing the chance of deleterious transposon insertions in the germline. A third possibility is suggested by a related system of RNAi and heterochromatin formation in fission yeast, in which degradation products from random abundant transcripts are used to prime Argonaute proteins and start a positive feedback loop [31
Aside from the piRNAs that are derived from repetitive elements and involved in the Ping-Pong mechanism, there are classes of piRNAs that are not repetitive. For example, the piRNA populations expressed at different stages of mammalian testis development are distinct and those found at the pachytene stage are depleted in repetitive sequences [32
]. In addition, some piRNAs are found in genes and are assumed to repress their host transcripts [33
]. Finally, there is some evidence that piRNAs are functional in the brain in rat [34
]. The connection between neural expression of piRNAs and the expression of transposable elements in the mammalian brain [35
] has been observed and is clearly intriguing, but there is currently no evidence to further connect these two aspects of neuroscience. In the rest of this review, we will focus on the repetitive piRNAs that are involved in the Ping-Pong mechanism and repress transposable elements because they are much better understood than the nonrepetitive piRNAs.
Overview of piRNA evolution
The piRNA system is known to be ancient as Piwi proteins, and the Ping-Pong signature are conserved in basal metazoans [36
]. However, no Piwi homologs have been found outside animals so the piRNA system appears to be an animal-specific innovation. Between closely related species, the genomic locations of many piRNA clusters are conserved, but the sequences of the piRNAs themselves are not conserved between rat and mouse [37
and C. briggsae
] or Drosophila melanogaster
and D. simulans
]. Thus, the overall picture of piRNA evolution at the sequence level is one of very rapid evolution.
A recent study of human piRNAs by one of the authors suggested that there is strong negative selection at the sequence level for human piRNAs but only in the three African populations and not any of the eight non-African populations studied [39
]. This observation is consistent with a recent report that African populations have much higher rates of transposon insertion than other populations [40
]. A further intriguing observation from the analysis of human piRNAs and transposable elements is the depletion of piRNA matches in the reverse transcriptase region of human LINE-1 elements, though not mouse LINE-1 elements [39
]. This observation suggests the possibility that at least one reverse transcriptase might be functional for the host and therefore protected from piRNA-mediated repression.
Beyond sequence divergence, it is also interesting to study the relationship of piRNA clusters and copy number changes, as an increase in copy number could potentially increase the level of gene expression of piRNAs. Assis and Kondrashov studied the evolution of piRNA clusters between mouse and rat and found a very high rate of piRNA cluster duplication, which they suggested is indicative of positive selection for higher expression level of piRNAs [37
Although the piRNA system is not understood well enough for detailed mathematical modeling, there has been one attempt by Lu and Clark [41
] at modeling piRNA-transposable element co-evolution using computer simulations. From their simulation, they suggested that retrotransposon insertions that are repressed by piRNAs can reach high frequencies or even be fixed in the population because their deleterious effect is attenuated by piRNA repression.
The idea that the piRNA pathway and transposable elements might co-evolve in a Red Queen-like scenario has been explored by a number of authors. In this scenario, alternating rounds of adaptation and counter-adaptation would lead to increased rates of positive selection. In a molecular evolution analysis examining species across the Drosophila
genus, it was found that a higher transposable element abundance is positively correlated with greater codon bias in piRNA pathway genes but not an increased rate of amino acid substitution in these genes [42
]. The authors suggested that these observations indicate that positive selection on piRNA pathway genes occurs mainly at the level of translation efficiency mediated by codon usage (although other explanations for codon bias are possible) as opposed to amino acid substitution [42
]. Further, a resequencing study of a number of defense genes in D. melanogaster and D. simulans
concluded that RNAi genes have the highest rate of adaptive evolution over all immune-system genes [43
]. Subsequent studies also found recurrent adaptation across the twelve sequenced Drosophila
genomes for a number of piRNA pathway genes, including SPN-E, AUB, KRIMP, SQU and ZUC [44
], as well as Rhino [45
]. Overall, these studies are consistent with elevated rates of evolution on piRNA pathway genes, consistent with its role in genome defense. While the molecular details of the Red Queen scenario for piRNAs and transposable elements are unclear, certain aspects of transposable element evolution, such as a higher global transposition rate, could select for certain features of piRNA-pathway genes, such as stronger binding affinity of the proteins for piRNAs.
PiRNAs and phenotypic capacitors
An interesting and somewhat contentious aspect of the role of the piRNA system in evolution is its role in canalization. Canalization, most famously associated with Waddington [46
], refers to the buffering of genetic or environmental insults to ensure developmental robustness. In a seminal paper, Rutherford and Lindquist [47
] suggested that Hsp90, a protein chaperone, is a phenotypic capacitor in Drosophila
, meaning that it buffers genetic variation but when it is compromised, that variation is revealed in multiple mutant phenotypes, at least some of which could be adaptive in certain environments [48
]. Similar results were subsequently demonstrated in Arabidopsis
], suggesting that Hsp90 might play an evolutionarily conserved role as a phenotypic capacitor.
The connection between canalization and the piRNA system comes from a recent report that in Drosophila
, Hsp90 regulates the piRNA pathway, which in turn regulates the insertion of transposons [50
]. It was further suggested that Hsp90 interacts in a protein complex with Piwi protein and mediates canalization by epigenetic silencing of genetic variation and suppressing transposon insertion [51
]. Thus, one potential mechanism by which the disruption of Hsp90 creates phenotypic variation is not by revealing previously cryptic variation as suggested by Rutherford and Lindquist but rather through de novo
mutations generated by transposon insertions. For this to be true, a strong bias in the preference in genome position for transposition insertion dependent on genetic background is required, and while such a preference is known to exist, it is not clear if it is strong enough to fully explain the results of the Rutherford and Lindquist experiments. Also, the piRNA study [51
] showed an effect on gene regulation separable from the effect on transposons. Conversely, imprecise transposon deletions could have a mutagenic effect and would necessarily be in the same place in the genome so more work needs to be done to define the exact role of piRNAs in canalization.