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Numéro
Med Sci (Paris)
Volume 29, Numéro 5, Mai 2013
Page(s) 487 - 494
Section Traduction
Publié en ligne 8 octobre 2014

© 2013 médecine/sciences – Inserm

Transposable elements, a threat for the genome

The genome is constantly threatened by a number of external events such as physical aggressions or exposure to mutagenic factors (ionising radiation or toxic substances) that cause mutations and substantial DNA rearrangements. There is also an intrinsic danger from the genome: mobile repeated sequences called transposable elements. These elements are DNA transposons and retrotransposons that move to other locations in the genome via cut-paste and copy-paste mechanisms, respectively, with potentially harmful consequences for the genome [1]. These potentially dangerous transposable elements occupy almost half of the human genome. Their large number and mobility have conferred an inestimable malleability to the remodelling of genomes in the course of evolution [2]. Even though most of these transposable elements are inactive (state of fossil DNA) because they have been altered by mutations, some remain active or can be reactivated. For example, class LINE1 retrotransposons (long interspersed elements) of which there are about 500,000 copies in mammalian genomes, account for 100 and 3,000 active copies in humans and mice, respectively [2], and some of them are implicated in diseases [3].

Several defence systems act against the harmful action and expansion of these transposable elements. In mammals, DNA methylation and histones modifications are indispensable for inhibiting the expression of transposable elements. This epigenetic inactivation is then transmitted to daughter cells. Another level of control is ensured by small RNAs containing between 20 and 30 nucleotides (nt). These small RNAs appear to play a very important role in germlines. The germline (or sexual line) transmits genetic information to the next generation. As any genetic alteration of these cells might be transmitted to descendants, it is of utmost importance to guarantee the genome stability of germlines, more than that of somatic (non-sexual) lines for the benefit of individuals and their species. In addition, there is a critical window in mammals, at the time of the formation and differentiation of primordial germ cells in the late embryogenesis, during which the epigenome (status of DNA methylation and histones modifications) of germ cells is remodelled. This renders these cells vulnerable to invasion by transposable elements. During this period, the genome is protected thanks to the presence of the special defence system based on small RNAs called piRNAs (PIWI-interacting RNAs) [47, 38, 39], which action can be compared to that of an immune system of the genome. This review summarises current knowledge and understanding of this class of RNA that began to be characterised only in 2006.

Characteristics of piRNAs

Until now, piRNAs have been detected in the germlines of all studied animals and seem to have appeared very early in the course of evolution, in an ancestor common to all animals [8]. It is strongly believed that they are major actors in the war against transposable elements during the gametogenesis of animals. It is to be noted that another class of endogenous small RNAs, endo-siRNA (endogenous small interfering RNA) also appears to be involved in the repression of transposable elements and seems to be very active in somatic cells [9, 10]. Even though piRNAs are similar to siRNAs and microRNAs (miRNA), they differ in several aspects [11]. The size of piRNAs is ranging between 21 and 30 nt, compared to about 21 nt for siRNAs and about 22 nt for miRNAs. These three RNA classes interact with two sub-families of Argonaut proteins: AGO and PIWI. AGO proteins interact with miRNAs and siRNAs [39], whereas PIWI proteins bind piRNAs. In mammals, although there are about a thousand unique sequences of miRNA, there are more than one million for piRNAs. In contrast to miRNAs, the sequences of piRNAs are conserved between species to only a very small extent and only their relative positions in the genome (synteny) appears to have been conserved. Finally, the biogenesis of piRNAs differs significantly from that of miRNAs and siRNAs [3941].

Biogenesis of piRNAs

Whereas miRNAs require maturation from characteristic stem-loop structures and siRNA need double-stranded RNA precursors [40, 41], piRNAs appear to arise from long single-stranded RNA precursors via a pathway that remains to be elucidated [11, 12]. Results of recent sequencing have shown that millions of different piRNAs arise from a limited number of loci, about two hundred in mammals, in which most are clustered. The piRNAs in these clusters arise from one of the two strands [4, 5]. Most of these clusters are located in regions rich in repeats or in intergenic regions. Their origin may in fact vary in different piRNA sub-populations specific to a given stage of differentiation. For example, in mammals there are piRNA populations associated with prepachytene or pachytene stages.

It is also to be noted that two to four different PIWI proteins have been systematically identified in animals and interact with piRNAs of different sizes and types. There are several aspects of the biogenesis of piRNAs that remain to be elucidated. Initially, an endonuclease, the best candidate is Zucchini, creates precursors named pre-piRNA (Figure 1) [13, 14]. In the worm C. elegans, an upstream sequence seems to recruit an endonuclease which is still to be uncovered [15] but in other species, it is not known what defines these regions as piRNA clusters. Pre-piRNAs are recognised by a PIWI protein that is responsible for a selection bias of candidates. Primary piRNAs indeed more often contain a U residue in 5’ position, the signature of the primary genesis of piRNAs. When the pre-piRNA binds the PIWI protein, it undergoes the action of a yet unidentified exonuclease that reduces its size to 21-30 nt [16]. The exact size of the piRNA is dependent on the bound PIWI protein. These primary piRNAs then undergo methylation of their 3’ end by HEN1 (HUA enhancer 1), a modification believed to protect them from premature degradation [17]. The primary piRNA can direct the PIWI protein to its target, e.g. mRNA of a retrotransposon, by total or partial basepairing. Since some of the PIWI proteins also have endonuclease activity, they cleave the target mRNAs at the 10th position from the 5’end of the bound piRNA, giving rise to the 5’ end of a new, secondary piRNA. As a result, the secondary piRNA contains two signatures: (i) its sequence is complementary to that of the primary piRNA over the first 10 nucleotides and (ii) since most of the primary piRNAs present a U residue at the first position, most of the secondary piRNAs present a A residue at position 10. This secondary piRNA can lead the same PIWI protein or another protein to cleave its target (transcripts from piRNA cluster), thereby creating a new piRNA called secondary because it is generated by PIWI proteins even though it remains similar to the primary piRNA. As only the piRNA target is cleaved, piRNAs can be reused by PIWI proteins to direct the cleavage to another target. This cycle amplifies the action of piRNAs. This amplification cycle partly or totally contributes to the biogenesis of piRNAs (see below) and is commonly called a ping-pong cycle [38].

thumbnail Figure 1.

Theoretical model of the primary biogenesis of piRNA and amplification via a ping-pong cycle. Zucchini endonuclease is thought to be responsible for the maturation of transcripts containing clusters of piRNA. Pre-piRNAs are recognised by a PIWI protein (Piwi A) with a preference for sequences with a uracil (U) in the first (5’) position. When associated with Piwi A, pre-piRNAs undergo maturation by a 3’-5’ exonuclease, as well as by HEN1 which adds a methyl group in 2’O of the 3’ end that makes them functional. The generated primary piRNAs can enter the ping-pong cycle. Through sequence complementarity, the primary piRNA directs the cleavage of the target retrotransposon (sense or antisense strand) thanks to the endonuclease activity brought by PIWI protein A. The newly generated 5’ end is recognised by another PIWI protein (Piwi B), but this protein can be the same as the first one. It is to be noted that the 3’ end of the secondary piRNA is matured and modified, just as the primary piRNA. The secondary piRNA can direct the cleavage activity of Piwi B on the transcript bearing piRNA clusters, thereby creating a new secondary piRNA which sequence is identical to that of the primary piRNA and so on. The piRNAs which 5’ end is formed by the action of Zucchini (or potentially another endonuclease) are called primary piRNAs and piRNAs which 5’ end is formed by PIWI proteins are called secondary piRNAs. piRNAs called A10 are secondary piRNAs while those called U1 may be primary or secondary.

Mechanisms of action of piRNAs

piRNAs silence transposable elements via different mechanisms, the most visible of which is the endonuclease activity of PIWI proteins. This enables them to cleave the target and therefore to trigger the degradation of the retrotransposons mRNA. Therefore, the system must generate a primary or secondary piRNA that can basepair with the target (antisense piRNA) in addition to the recruitment of a catalytically active PIWI protein. This last point can be examined by mutating the catalytic DDH triad of PIWI proteins and measuring the resulting effects on populations of piRNA containing the above-mentioned signatures [18, 19].

Mechanism of action of piRNAs in mice

There are three PIWI proteins in mice, called MILI, MIWI and MIWI2. Only MILI is expressed in both sexes. In male, MILI is expressed from the embryonic until the round spermatids stage, MIWI2 is detected from the embryonic stage up to three days after birth, and MIWI is expressed postnatal at the stage of pachytene spermatocytes and round spermatids (Figure 2). In the course of spermatogenesis, these three PIWI proteins have different functions according to the prepachytene and pachytene piRNA populations with which they are associated [39]. In the prepachytene population of piRNAs, primary piRNAs bind MILI which generates secondary piRNAs which then bind MIWI2 after cleavage of the target [18, 20, 21]. MIWI2 moves within the nucleus and guides DNA methylation of transposable elements promoters [18, 21]. As MIWI2 has no endonuclease activity (in contrast to MILI and MIWI), there is no ping-pong cycle between MILI and MIWI2 [18]. It is nevertheless to be noted that a ping-pong amplification is seen between two MILI proteins. In summary, a transfer is created between MILI that degrades transposons mRNAs and can amplify the signal by a ping-pong cycle, and MIWI2 that will have a long term influence on the expression of this transposon because the daughter cells will inherit epigenetic marks: DNA methylations as well as histone modifications [43]. There are thus two types of responses to the expression of a retrotransposon at the prepachytene stage: rapid post-transcriptional repression by MILI and epigenetic repression by MIWI2 coordinated with MILI. The reaction involving the piRNA machinery is therefore adapted, rapid, amplified and memorised, reminding the action of the immune system. At the postnatal stage, pachytene piRNAs bind both MILI and MIWI displaying both endonuclease activity, thereby repressing any retrotranposons that would have escaped the first wave of repression [19]. MIWI exerts a watchdog function, but its direct interaction with mRNAs suggests a piRNA independent role in mRNA regulation [22]. It is also to be noted that MILI and MIWI interact with the translation machinery, suggesting a possible role in translational repression [23].

thumbnail Figure 2.

Role of piRNAs in the inactivation of retrotransposons in mice. A. Expression of PIWI proteins and piRNAs during spermatogenesis in mice. After their migration to gonads, primordial germ cells multiply until cessation around 15 dpc (days post-coitus). This is followed by de novo methylation of DNA. Spermatogonia resumes divisions at 3 dpp (days post-partum) and meiosis starts at 10 dpp. The first round haploid spermatids appears at 14 dpp. B. Biogenesis and function of pachytene piRNAs. After leaving the nucleus, the transcripts harboring piRNA clusters generate primary piRNAs which recruit MILI. The endonuclease activity of MILI cleaves its target to give rise to a secondary piRNA. A ping-pong cycle between two MILI proteins may amplify the production of secondary piRNAs. The secondary piRNAs which bind MIWI2 can enter the nucleus and control the DNA methylation of retrotransposons by basepairing with a transcript in course of elongation to recruit an unknown mechanism leading to histone modification and DNA methylation.

Mechanism of action of piRNAs in C. elegans

A recent study in the worm C. elegans has revealed a novel mechanism of action [24, 25]. The population of primary piRNAs (21U-piRNA) guides a PIWI protein, PRG-1, in contact with a transcript corresponding to a retrotransposon or a foreign element (Figure 3). PRG-1 exerts no endonuclease activity, but recruits an RNA-dependent RNA-polymerase complex (RdRP) that uses the target transcript as template to generate small guide RNAs called 22G-RNAs. These latter guides recruit the Argonaut protein WAGO9, known for its involvement in the siRNA system, to repress the retrotransposon by cleavage and by epigenetic control. The fact that one piRNA can lead to several 22G-RNAs is also a signal amplification step. This work also showed that basepairing between the piRNA and the target must be imperfect, which considerably increases the number of potential targets. It is noteworthy that the evolution of piRNAs sequences would prevent targeting its own protein coding mRNAs. An alternative explanation might be the involvement of the Argonaut protein CSR-1, also associated with small RNAs. CSR-1, could recognise protein coding mRNAs through small RNA basepairing and thus spare them from degradation [25] (Figure 3). Finally, the repression initiated by piRNAs appears to be maintained over several generations of worms [26-43]. In summary, this distinction between self and nonself elements is reminding the mechanisms of innate natural immunity.

thumbnail Figure 3.

Model of the role of piRNAs in the distinction between self and nonself RNAs in the worm C. elegans. In germlines, a piRNA complex associated with PRG-1 inspects RNAs to identify targets with imperfect complementarity. A system based on the use of protein CSR-1 and endo-siRNA protects cellular mRNAs (1a). Recognition of the target, a transposable element or a foreign element by a piRNA (1b) recruits an RNA-dependent RNA polymerase (RdRP) (2). This enzyme generates many small RNA guides called 22G-RNA (3) which bind WAGO9, either to amplify the signal by recruiting RdRP and generating new 22G-RNA, or to inactivate the transposon. There are two mechanisms of repression that operate, in the nucleus by epigenetic modifications (4a) and in the cytoplasm by degradation of the target (4b).

Mechanism of action of piRNAs in Drosophila

In Drosophila, primary antisense piRNAs are generated from copies of transposable elements called “defective” because they are partly inactivated owing to the accumulation of mutations and deletions. A ping-pong amplification cycle enables primary antisense piRNAs to target copies of functional transposable elements to produce sense piRNAs that in turn can guide PIWI proteins to copies of defective transposable elements to produce antisense secondary piRNAs [12]. Primary piRNAs, however, are not the only piRNAs that can trigger the ping-pong amplification cycle. The study of I-element, a retrotransposon expressed within the germline of Drosophila ovaries, shows that this amplification cycle can be triggered by piRNAs deposited in the embryo. In the course of its evolution, D. melanogaster has undergone two waves of invasion by the I-elements. This explains the existence of R (reactive) strains having undergone an ancient invasion and containing only vestiges of I-elements, and I (inducer) strains having been subjected to a second, more recent invasion. Crossing R females with I males results in sterile offspring which gonads are dystrophic, while crossing I females with R males results in fertile offspring. This phenomenon, called hybrid dysgenesis, remained unexplained for a long time, until the discovery of piRNAs. This results from the fact that maternal piRNAs are deposited in the embryo and can initiate an amplification cycle enabling germlines of the next generation to defend themselves against I-elements [27]. I females thus transmit sufficient piRNAs specific against I-element to their offspring to inhibit its expression, while the quantity of piRNAs transmitted by R females is insufficient to induce its inhibition. Eggs laid by old R females, however, contain more piRNA than those laid by young R females [28]. As an R female ages, it can therefore acquire the capacity to repress the I-element, even from copies of defective transposable elements, a capacity that can in turn be transmitted from mother to daughter by depositing piRNA in the embryo. piRNAs can therefore transmit epigenetic information over several generations.

Elements of the piRNA machinery are confined in cytoplasmic bodies

Genetic studies have shown that, in addition to PIWI proteins, many other factors are indispensable for the piRNA machinery. The piRNA machinery in fact requires the formation of a large complex comparable to the RISC complex (RNA-induced silencing complex) associated with miRNAs [11, 29, 39]. PIWI proteins differ from AGO proteins by the presence of methylations marks at arginine residues that recruit gonad-specific proteins displaying Tudor domains which are an assembly platform for a specific piRISC complex [42]. One difference from the miRISC complex is that elements of the piRNA machinery are confined to cytoplasmic granules seen at certain stages of differentiation of germlines [30]. In mammals, most elements of the piRNA machinery are found in three different structures: intermitochondrial cements, processing bodies (P bodies) and chromatoid bodies. The latter are perinuclear, contain the largest number of these factors and undergo remodelling during spermatogenesis [31]. The role of these membrane-less aggregates, however, remains unknown. They could play a role in the degradation of piRNA targets or in the degradation of piRNAs and associated proteins, but also in the storage of piRNAs for future use. Sequestering the components of this machinery in different structures limits their action to a precise time window and prevents interference with mechanisms involving other small RNAs. This additional level of regulation remains poorly defined and requires further work.

Defects of the piRNA machinery and male sterility

Surprisingly, in mouse, until now, all mutations resulting in a loss of function of the major piRNA machinery components, lead to male sterility by interrupting spermatogenesis at different stages [29]. Even though females also express a part of the elements of the piRNA machinery, they are unaffected for an unclear reason. It has been suggested that a system based on the use of endo-siRNA could compensate the absence of a part of the piRNA machinery [32, 33]. This class of endo-siRNA could reinforce the repression implemented by piRNAs and is emphasizing that other classes of small RNAs are participating in the repression of transposable elements [34]. In males, any failure of the piRNA machinery results in severe degeneration of germ cells. Thus, it would be relevant to determine the potential involvement of the piRNA machinery in human male sterility. For example, one study involving 490 azoospermia or oligospermia patients has identified nine marks of single nucleotide polymorphism (SNP) in PIWI proteins [35]. In addition, overexpression of HIWI, the human equivalent of MIWI, is related to certain cancers [36], suggesting a possible involvement of PIWI proteins in tumorigenesis [37]. Furthermore, the expression of piRNAs or PIWI proteins occasionally observed in somatic cells could lead to the conservation of certain properties of stem cells [37]. In summary, it is presently difficult to measure the entire scope of consequences of a piRNA machinery defect even though its role in germ cells differentiation has been clearly established.

Conclusion

The investigations of the piRNA machinery have been limited for a long time because of its restricted expression to germ cells and because of the diversity of piRNAs. Indeed more than one million unique sequences exist, each weakly expressed and selected by mechanisms that are still unclear. The use of next generation sequencing techniques and the use of three study models (mouse, C. elegans and Drosophila) have enabled us to understand their crucial role in the inactivation of transposable elements. This aspect is very clear during a phase of gametogenesis during which the epigenetic code is remodelled, but also during the reactivation of transposable elements or during contacts with vectors of these transposable elements such as retroviruses. In that sense, the piRNA machinery can be seen as an immune system at the scale of the genome. Akin to natural immunity, piRNAs are the first barrier against active copies of retrotransposons carried by the genome. The piRNA machinery also differentiates self and nonself by mechanisms, not yet completely elucidated, which are based in particular on other small RNAs such as endo-siRNAs. In this framework, piRNAs guide a complex bringing endonuclease activity to the target to degrade. An amplification step ensures the efficiency of its action. In addition, similar to acquired immunity and clonal expansion observed with antibody-producing lymphocytes, the piRNA machinery offers a large repertoire of piRNAs. Beyond this sequence variability, the possibility of mismatches allows a large number of targets. In summary, only piRNAs that recognise their target are amplified. In this context, in addition to posttranscriptional repression, piRNAs direct DNA methylation and histone modifications in the nucleus. This epigenetic control keeps memory of the inactivation of a sequence that is now inserted in the genome of a given cell line, and also over several generations of offspring. Finally, the fact that piRNAs target elements other than transposable elements, such as genes for proteins or non-coding RNAs, as well as the enigmatic presence of the piRNA machinery in somatic cells (in particular in cancer cells), are harbingers of many experimental surprises to come.

Conflict of interest

The authors state that there is no conflict of interest concerning the data published in this article.

Acknowledgments

S. Muller was supported by the ARC (French Association for Cancer Research) and R.R. Pandey was supported by the EMBO (European Molecular Biology Organization). We apologize to colleagues whose work could not be cited for reasons of space restrictions.

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All Figures

thumbnail Figure 1.

Theoretical model of the primary biogenesis of piRNA and amplification via a ping-pong cycle. Zucchini endonuclease is thought to be responsible for the maturation of transcripts containing clusters of piRNA. Pre-piRNAs are recognised by a PIWI protein (Piwi A) with a preference for sequences with a uracil (U) in the first (5’) position. When associated with Piwi A, pre-piRNAs undergo maturation by a 3’-5’ exonuclease, as well as by HEN1 which adds a methyl group in 2’O of the 3’ end that makes them functional. The generated primary piRNAs can enter the ping-pong cycle. Through sequence complementarity, the primary piRNA directs the cleavage of the target retrotransposon (sense or antisense strand) thanks to the endonuclease activity brought by PIWI protein A. The newly generated 5’ end is recognised by another PIWI protein (Piwi B), but this protein can be the same as the first one. It is to be noted that the 3’ end of the secondary piRNA is matured and modified, just as the primary piRNA. The secondary piRNA can direct the cleavage activity of Piwi B on the transcript bearing piRNA clusters, thereby creating a new secondary piRNA which sequence is identical to that of the primary piRNA and so on. The piRNAs which 5’ end is formed by the action of Zucchini (or potentially another endonuclease) are called primary piRNAs and piRNAs which 5’ end is formed by PIWI proteins are called secondary piRNAs. piRNAs called A10 are secondary piRNAs while those called U1 may be primary or secondary.

In the text
thumbnail Figure 2.

Role of piRNAs in the inactivation of retrotransposons in mice. A. Expression of PIWI proteins and piRNAs during spermatogenesis in mice. After their migration to gonads, primordial germ cells multiply until cessation around 15 dpc (days post-coitus). This is followed by de novo methylation of DNA. Spermatogonia resumes divisions at 3 dpp (days post-partum) and meiosis starts at 10 dpp. The first round haploid spermatids appears at 14 dpp. B. Biogenesis and function of pachytene piRNAs. After leaving the nucleus, the transcripts harboring piRNA clusters generate primary piRNAs which recruit MILI. The endonuclease activity of MILI cleaves its target to give rise to a secondary piRNA. A ping-pong cycle between two MILI proteins may amplify the production of secondary piRNAs. The secondary piRNAs which bind MIWI2 can enter the nucleus and control the DNA methylation of retrotransposons by basepairing with a transcript in course of elongation to recruit an unknown mechanism leading to histone modification and DNA methylation.

In the text
thumbnail Figure 3.

Model of the role of piRNAs in the distinction between self and nonself RNAs in the worm C. elegans. In germlines, a piRNA complex associated with PRG-1 inspects RNAs to identify targets with imperfect complementarity. A system based on the use of protein CSR-1 and endo-siRNA protects cellular mRNAs (1a). Recognition of the target, a transposable element or a foreign element by a piRNA (1b) recruits an RNA-dependent RNA polymerase (RdRP) (2). This enzyme generates many small RNA guides called 22G-RNA (3) which bind WAGO9, either to amplify the signal by recruiting RdRP and generating new 22G-RNA, or to inactivate the transposon. There are two mechanisms of repression that operate, in the nucleus by epigenetic modifications (4a) and in the cytoplasm by degradation of the target (4b).

In the text