shutdown: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

Gene name - shutdown

Synonyms -

Cytological map position - 59D8--60A2

Function - immunophilin, enzyme

Keywords - piRNA biogenesis, oogenesis, spermatogenesis

Symbol - shu

FlyBase ID: FBgn0003401

Genetic map position - chr2R:19728984-19730802

Classification - FKBP-type peptidyl-prolyl cis-trans isomerases 1

Cellular location - cytoplasmic and nuclear



NCBI links: Precomputed BLAST | Entrez Gene | UniGene
BIOLOGICAL OVERVIEW

In animal gonads, PIWI proteins and their bound 23-30 nt piRNAs guard genome integrity by the sequence specific silencing of transposons. Two branches of piRNA biogenesis, namely primary processing and ping-pong amplification, have been proposed. Despite an overall conceptual understanding of piRNA biogenesis, identity and/or function of the involved players are largely unknown. This study demonstrates an essential role for the female sterility gene shutdown in piRNA biology. Shutdown, an evolutionarily conserved cochaperone collaborates with Hsp90 during piRNA biogenesis, potentially at the loading step of RNAs into PIWI proteins. Shutdown is shown to be essential for both primary and secondary piRNA populations in Drosophila. An extension of this study to previously described piRNA pathway members revealed three distinct groups of biogenesis factors. Together with data on how PIWI proteins are wired into primary and secondary processing, a unified model for piRNA biogenesis is proposed (Olivieri, 2012).

PIWI interacting RNAs (piRNAs) are a class of animal small RNAs. They are bound by PIWI family proteins and guide the sequence specific silencing of selfish genetic elements such as transposable elements (TEs). Defects in the piRNA pathway lead to TE derepression, genomic instability and ultimately sterility (Olivieri, 2012).

In Drosophila, most piRNAs are generated from two sources; on the one hand, these are piRNA cluster transcripts that originate from discrete genomic loci and serve as reservoirs of TE sequences; on the other hand, these are RNAs derived from active TEs that engage - together with cluster transcripts - in a piRNA amplification loop called the ping-pong cycle (Olivieri, 2012).

Two modes of piRNA biogenesis exist: (1) during primary piRNA biogenesis, a single stranded RNA is processed into pre-piRNAs, which are loaded onto PIWI proteins and are subsequently 3' trimmed and methylated, yielding mature piRNA induced silencing complexes (piRISCs). (2) piRISCs with active slicer activity can trigger secondary piRNA biogenesis, where a new piRNA is formed out of the sliced target RNA. In the presence of corresponding sense and antisense precursor RNAs, secondary piRNA biogenesis acts as the ping-pong amplification loop. The two piRNAs engaged in ping-pong have opposite orientation and exhibit a characteristic ten nucleotide 5' overlap (ping-pong signature) (Olivieri, 2012).

Primary and secondary piRNA biogenesis co-occur in germline cells, complicating the genetic and mechanistic dissection of these processes. However, somatic cells of the gonad also harbor a piRNA pathway and this is based exclusively on primary piRNA biogenesis. The Drosophila ovary is therefore ideally suited to identify and characterize factors required for either primary or secondary piRNA biogenesis or both (Olivieri, 2012).

Somatic support cells of the Drosophila ovary express Piwi as the only PIWI family protein. Primary piRNA biogenesis is thought to take place in peri-nuclear Yb-bodies, where the RNA helicases Armitage (Armi) and Yb as well as the TUDOR domain protein Vreteno (Vret) accumulate. In addition to these three factors, the putative mitochondria-localized nuclease Zucchini (Zuc) and the RNA helicase Sister of Yb (SoYb) are essential for piRNA biogenesis in the soma. Formation of mature Piwi-RISC triggers its nuclear import, while failure in piRNA biogenesis results in destabilization of presumably unloaded Piwi. Mature Piwi-RISC triggers TE silencing by an unknown mechanism that requires Piwi's nuclear localization but not its slicer activity (Olivieri, 2012).

With the exception of Yb, the above mentioned biogenesis factors are also essential in germline cells for the formation of Piwi-RISC. Germline cells, however, express two additional PIWI proteins, Aubergine (Aub) and Argonaute 3 (AGO3), which localize to the cytoplasm and are enriched in peri-nuclear nuage. Aub and AGO3 are the main players in the ping-pong cycle. Several factors with essential or modulatory roles in the ping-pong cycle have been identified. These are the RNA helicases Spindle-E and Vasa and the TUDOR domain proteins Krimper, Tejas (Tej), Qin and Tudor (Olivieri, 2012).

The analysis of piRNA populations from wild-type and piRNA pathway mutant ovaries indicated that Piwi is mainly a recipient of primary piRNAs, while Aub and AGO3 are predominantly or exclusively recipients of secondary piRNA biogenesis. Given this, three major questions arise: (1) Are primary and secondary piRNA biogenesis processes genetically and mechanistically separate or do common factors act in both processes? (2) In which processing step do identified piRNA biogenesis factors act? (3) How are the three PIWI family proteins wired into piRNA biogenesis? In other words, are certain PIWI proteins only receiving primary or only secondary piRNAs (Olivieri, 2012)?

This study shows that the female sterility gene shutdown (Munn, 2000; Schüpbach and Wieschaus, 1991) encodes a piRNA biogenesis factor. Shu is required for all piRNA populations in ovaries and it acts downstream of known piRNA biogenesis factors. A comparison of Shu to several other pathway factors led to the definition of three major groups of piRNA biogenesis factors. In combination with data on how PIWI proteins are wired into piRNA biogenesis, a model is proposed that accounts for the distinct association of piRNA subpopulations with specific PIWI proteins in Drosophila (Olivieri, 2012).

The outcome of this work is threefold: (1) The cochaperone Shutdown is essential for the biogenesis of all Drosophila piRNA populations. (2) Three major groups of piRNA biogenesis factors can be distinguished. (3) Piwi and Aub but not AGO3 are loaded with primary piRNAs, explaining how the cell maintains highly specific piRNA populations in the three PIWI proteins (Olivieri, 2012).

A remarkable feature of the shu mutant phenotype is that piRNA populations for every TE collapse. This already points to a common piRNA biogenesis step downstream of the primary and secondary branches. Epistatic analysis in somatic follicle cells is consistent with Shu acting at a late step in piRNA biogenesis: Shu is not required for the localization of any known biogenesis factor to Yb-bodies. On the other hand, Shu's localization to Yb-bodies depends on all other biogenesis factors and even on Piwi, arguing that unloaded Piwi recruits Shu to the Yb-body. Similarly, Shu colocalizes with nonloadable AGO3 in OSCs as well as in ovaries defective of ping-pong in discrete foci that also contain and are dependent on Krimp. Thus, in wild-type and in biogenesis mutants, Shu appears to colocalize with unloaded PIWI proteins (Olivieri, 2012).

Shu's C-terminal TPR domain falls into the class of Hsp90 binders and Hsp90 is important for small RNA loading into Argonaute proteins (Iki, 2010; Iwasaki, 2010; Miyoshi, 2010). In addition, the plant cochaperone Cyp40 interacts with Hsp90 via its TPR domain and is a critical cofactor for small RNA loading into AGO1 (Iki, 2012). The genetic and localization data support an analogous role for Shu and Hsp90 during small RNA loading into PIWI proteins. Clearly, in vitro assays will be crucial to dissect the precise order of events and the molecular role of Shu, especially its PPIase domain (Olivieri, 2012).

A major challenge in the field is to assemble piRNA biogenesis factors into pathways that explain the stereotypic populations of piRNAs in vivo. Advantage was taken of efficient germline specific knockdowns to study the impact of several factors on piRNA populations. Based on levels and localization of PIWI proteins as well as on piRNA populations obtained from several pathway factor knockdowns, three major groups of piRNA biogenesis factors are proposed (Olivieri, 2012).

Group I factors are required for primary piRNA biogenesis but dispensable for secondary biogenesis. In fact, piRNAs that initiated ping-pong in group I knockdowns were amplified and ping-pong signatures of such TEs were strongly increased, presumably as primary piRNAs that do not feed into ping-pong were absent (Olivieri, 2012).

Group II factors are specific for ping-pong, as primary piRNA biogenesis feeding into Piwi was unaffected. An alternative explanation that cannot be excluded is that some or all group II genes are required specifically for Aub biology (primary and secondary) per se. This would similarly leave Piwi bound piRNAs intact and would lead to a collapse in ping-pong. Given the data on Aub loading in OSCs, a model is favored however where the primary biogenesis machineries that feed Aub and Piwi are very similar (Olivieri, 2012).

Finally, group III factors are required for the biogenesis of Piwi/Aub/AGO3 bound piRNAs. The prototypic member of this group is Shu. Loss of Shu affects essentially all piRNA populations to the same extent. It is noted that analysis of piRNA populations from vret mutants indicated a role for this group III factor in primary biogenesis but not ping-pong (Handler, 2011; Zamparini, 2011). The distorted tissue composition of vret mutant ovaries coupled with perdurance of maternal Vret protein or RNA may underlie this discrepancy. The existence of group III factors predicts that primary and secondary piRNA biogenesis feed into a final piRISC maturation step that requires a set of common factors for all PIWI proteins. Given that piRNA biogenesis -- irrespective of the source of the precursor RNA -- requires an RNA loading step as well as a 3' trimming step, the existence of group III factors suggests itself (Olivieri, 2012).

The three proposed groups serve as a rough classification of biogenesis factors. Clearly, at a molecular level, the precise role of each factor within the biogenesis process will vary considerably. Of note, the classification of group I and group II genes extends to the mouse piRNA pathway. The Armi and Zuc orthologs MOV10L1 and PLD6 are required for primary piRNA biogenesis, whereas mouse VAS and TDRD9 (mouse Spn-E) were reported to be dispensable for primary biogenesis but are required for secondary biogenesis pathway (Olivieri, 2012).

The data indicate that Aub is not only loaded via ping-pong, but also via primary piRNA biogenesis. It is also postulate that Aub and Piwi proteins are wired into primary piRNA biogenesis processes in a very similar manner, meaning that they require the same or highly overlapping core factors (e.g., Armi or Zuc). In agreement with this, ectopically expressed Aub is loaded in OSCs that harbor a fully functional primary pathway but lack critical ping-pong factors such as Vas. The genetic requirements for Aub loading in OSCs are identical to those for Piwi. It is extrapolated from this that the core primary biogenesis machinery that loads Piwi in the soma also loads Piwi and Aub in the germline. Analyses of piRNA populations from armi versus piwi or aub-GLKDs support a model where Armi and Zuc are required for the biogenesis of both Piwi and Aub bound primary piRNAs. The possibility is not excluded that - despite a similar biogenesis machinery - populations of primary piRNAs in Aub and Piwi are different. For example, differences in subcellular localizations of PIWI proteins as well as piRNA precursor RNAs might result in such differences (Olivieri, 2012).

In contrast to Aub, AGO3 was unstable in OSCs. Coexpression of Aub or simultaneous knockdown of krimp had no impact on AGO3 stability. It is therefore concluded that primary piRNA biogenesis is incompatible with AGO3. In fact, also in the germline genetic data indicated that AGO3 depends on secondary piRNA biogenesis for being loaded. Blocking AGO3's access to the primary biogenesis machinery would allow the cell to load AGO3 with a unique class of piRNAs if it couples AGO3 loading to a precursor RNA originating from Aub-slicer mediated cleavage of an active TE. This would explain the remarkable bias of AGO3 bound piRNAs being sense and carrying an Adenosine at position ten (Olivieri, 2012).

Interestingly, on a primary sequence level Aub -- despite its significantly different biology -- is more closely related to Piwi than to AGO3. A critical question emanating from this is to which extent Piwi is participating in ping-pong, and if it does not, why. A weak, yet statistically significant, ping-pong signature has been observed between Piwi and AGO3 bound piRNAs. This could mean that there is indeed low level of Piwi-AGO3 ping-pong. An alternative explanation is that the Piwi-AGO3 signal is a misleading computational signal: If Piwi and Aub are loaded via the same primary biogenesis machinery, initiator piRNAs for ping-pong that end up in Aub also end up in Piwi. As primary piRNA biogenesis appears to be nonrandom and preferentially processed piRNAs likely trigger ping-pong more robustly, an 'artificial' AGO3/Piwi ping-pong signature might result (Olivieri, 2012).

What could be the molecular basis of why Piwi does not or only moderately participate in ping-pong? Either, specific features of Aub (e.g., symmetric Arginine methylation) are funneling this protein into ping-pong and similar features are absent on Piwi. Or, the mere sequestration of Piwi into the nucleus prevents Piwi from participating in ping-pong. Notably, N-terminally truncated Piwi that is still loaded but that cannot translocate into the nucleus is enriched in nuage the proposed site of secondary piRNA biogenesis. A simple difference in the subcellular localization of Aub and Piwi might thus contribute to the dramatic differences of piRNA populations residing in Aub or Piwi (Olivieri, 2012).

shutdown encodes a novel FK506-binding protein essential for the formation of germline cysts during oogenesis

In Drosophila, the process of oogenesis is initiated with the asymmetric division of a germline stem cell. This division results in the self-renewal of the stem cell and the generation of a daughter cell that undergoes four successive mitotic divisions to produce a germline cyst of 16 cells. shutdown is essential for the normal function of the germline stem cells. Analysis of weak loss-of-function alleles confirms that shutdown is also required at later stages of oogenesis. Clonal analysis indicates that shutdown functions autonomously in the germline. shutdown was identified using a positional cloning approach. Consistent with its function, the RNA and protein are strongly expressed in the germline stem cells and in 16-cell cysts. The RNA is also present in the germ cells throughout embryogenesis. shutdown encodes a novel Drosophila protein similar to the heat-shock protein-binding immunophilins. Like immunophilins, Shutdown contains an FK506-binding protein domain and a tetratricopeptide repeat. In plants, high-molecular-weight immunophilins have been shown to regulate cell divisions in the root meristem in response to extracellular signals. These results suggest that shutdown may regulate germ cell divisions in the germarium (Munn, 2000).

In contrast to wild-type ovaries, 40%-60% of the ovarioles from newly eclosed shu mutant females do not contain any developing egg chambers. Usually, the other ovarioles contain only one to three egg chambers and their numbers fail to increase as the females age. None of the egg chambers develop into normal eggs but instead degenerate by mid- to late-oogenesis. Those egg chambers with the correct number of germ cells may have arisen through the differentiation of prestem cells, i.e., germ cells that developed as cystoblasts without first being established as stem cells. The strong cytoplasmic staining of germ cells with antibodies recognizing the Sex Lethal protein indicates that shu function is dispensable for the establishment of the female mode of sexual differentiation of the germ cells. To determine the developmental stage of shu mutant germ cells, they were analyzed using an antibody specific for the cytoplasmic form of the Bam protein. In wild-type germaria, the cytoplasmic form of Bam accumulates in mitotic cystoblasts and cystocytes. However, in newly eclosed shu mutant females no staining is observed. This suggests that, after the first few egg chambers are formed, no further mitotic cystoblasts develop. Instead, these germ cells appear to redivide to form clusters of abnormal germ cells that degenerate or occasionally produce tumorous cysts. Collectively, these phenotypes suggest that shu's function is essential for the normal activity of the stem cell. The absence of wild-type function could affect the first asymmetric division of the stem cell, resulting in an abnormal cystoblast that is compromised in its ability to undergo the normal four rounds of mitotic division. In addition, this abnormal asymmetric division would affect stem cell renewal, resulting in an abnormal stem cell that divides several times to produce ill-fated germ cell clusters (Munn, 2000).

Analysis of the shu phenotype, using a complete loss-of-function allele, has revealed requirements for shu in the normal behavior and differentiation of the germline. The few developing egg chambers with 16 germ cells present in newly eclosed shuWQ41 females have presumably arisen through the differentiation of prestem cells, i.e., germ cells that developed as cystoblasts without first being established as stem cells. These early egg chambers develop until midoogenesis and then degenerate. Germline stem cells are clearly established in the absence of shu function, because they express Sex Lethal protein and have spectrosomes. Although the stem cells appear to have undergone several divisions, their progeny do not become committed to the normal pathway of germline differentiation. Instead, they presumably redivide to produce clusters of ill-fated germ cells. This phenotype contrasts with that of mutants such as bam and bgcn, which specifically block cystoblast differentiation and result in tumors of mitotically active cells that continue to behave like stem cells. As shu females age, a loss of germ cells is observed, resulting in agametic ovarioles. This indicates that stem cell renewal is also affected by the loss of shu function. One way in which these phenotypes could have arisen is through an aberrant asymmetric division of the stem cell (Munn, 2000).

A similar asymmetric division controls the production of primary spermatocytes from male germline stem cells during spermatogenesis. Strong loss-of-function alleles of shu also result in male sterility. The mutant testes contain fewer than normal elongating sperm bundles: the apical tip of the testes, where the germline stem cells divide, appears reduced compared to wild type, and many of the cells are degenerating (Tirronen, 1993). These observations indicate that shu also has an essential function in germline stem cell regulation in males (Munn, 2000).

Mutations in decapentaplegic (dpp), pumilio (pum), nanos (nos), piwi, and fs(1)Yb that affect stem cell maintenance and asymmetric division have been described. While the source of the dpp signal remains unknown, the function of both piwi and fs(1)Yb is thought to be required in the somatic cells of the terminal filament. Both piwi and fs(1)Yb have been proposed to be required for the production of a somatic signal that regulates the germline stem cells. In contrast, pum and nos have been reported to function in the germline. The analysis of ovarian germline clones indicates that shu also functions in the germline autonomously. This is supported by the observation that both male and female sterility can be rescued using a transgene whose expression is controlled by the germline-specific otu promoter. As some of the phenotypes observed in shu are similar to those described for piwi and fs(1)Yb, it is possible that shu is required for the response of the germline cells to these somatic signals. No effects on fertility have been observed in transheterozygous combinations of shu with piwi, pum, or nos. In contrast to shu, mutations in dpp, piwi, pum, and nos allow the production of mature eggs. The other phenotypes observed in shu egg chambers, including a loss of oocyte identity and occasionally mispositioned oocytes, are therefore likely due to later requirements for shu in egg chamber development. Consistent with this possibility, shu expression increases in newly formed 16-cell cysts (Munn, 2000).

The expression pattern of the SHU mRNA suggests that shu may function in germline development during embryogenesis. The mRNA is incorporated into the pole cells and can be detected in the germ cells throughout their migration into the embryonic gonad. A number of RNAs have been identified that are incorporated into the pole cells including cyclin B, germ cell less, hsp83, nos, orb, oskar, pum, tudor, and vasa. Mutations in some of these genes also affect early germline development. Since the existing alleles of shu fail to produce normal eggs that are fertilized, the possible effects of zygotic shu function on germ cell migration or proliferation could not be studied (Munn, 2000).

The Shu protein shows significant homology to an evolutionarily conserved class of proteins, the immunophilins. Although these proteins have been shown, in vitro, to catalyze changes in protein folding, their function in vivo remains unclear. The best characterized of the FK506BPs is the low-molecular-weight immunophilin human FKBP12. It is a cytosolic protein that has been implicated in signal transduction (reviewed by Marks, 1996). In vertebrates, FKBP12 has also been proposed to regulate translation through its association with the FKBP12-associated rapamycin-binding protein (reviewed by Brown, 1996). This complex regulates the binding of translation initiation factors to the 5' end of the mRNA. The Drosophila gene vasa encodes a germline-specific homologue of the translation initiation factor eIF4A. Null alleles of vasa produce a variety of phenotypes that include atrophied germaria containing reduced numbers of developing germline cysts. No genetic evidence has been found that shu functions in this particular pathway, because females trans-heterozygous for a null allele of shu and a deficiency that removes vasa show no defects in oogenesis (Munn, 2000).

In addition to its FK506-binding domain, Shu contains a predicted TPR motif. This is a protein-protein interaction motif that was originally identified in cell cycle regulatory proteins but has since been found in a number of different proteins with no common biochemical function (Lamb, 1995). A mutation in an allele of shu within the TPR has been identified indicating the importance of this motif for shu function. This allele does not cause male sterility, suggesting that the protein might function differently in spermatogenesis, perhaps through interacting with different partners. Alternatively, it is also possible that spermatogenesis is less sensitive to reductions in levels of wild-type function (Munn, 2000).

The presence of a TPR motif, in addition to the FK506-binding domain, is characteristic of the high-molecular-weight immunophilins, an evolutionarily conserved family of proteins whose function remains uncharacterized. Interestingly, there are no examples of this type of immunophilin in yeast or Caenorhabditis elegans. Mammalian immunophilins were originally identified in complexes of HSP90 with unliganded steroid hormone receptors. It has been proposed that immunophilins function in the cytoplasmic to nuclear targeting of these complexes. Interestingly, the germline expression pattern of the Drosophila homolog of HSP90, Hsp83, is strikingly similar to that of shu (Ding, 1993). Biochemical experiments will confirm whether Shu interacts with HSP83 in the germline or with alternative partners (Munn, 2000).

Unfortunately, the lack of mutants in the mammalian immunophilins has prevented the identification of their in vivo functions. Human FKBP6 maps to a common 1-Mb deletion in patients with William's syndrome, a developmental disorder associated with a haploinsufficiency at chromosome 7q11.23. William's syndrome has multiple associated phenotypes. Together with the large size of the deletions, this has made it difficult to correlate specific gene functions with a particular aspect of the disease. Human FKBP6 and rodent FKBP52 are both expressed at particularly high levels in testes, suggesting that immunophilins may have a conserved function in germline development. Interestingly, germline stem cells of the mammalian testes, like Drosophila germline stem cells, undergo asymmetric, self-renewing divisions (Munn, 2000 and references therein).

Some insight into the function of immunophilins was recently obtained through the analysis of the pasticcino-1 (pas-1) mutant in A. thaliana. The pas-1 mutant was isolated in a screen for mutants that showed an abnormal response to the cell division-promoting plant hormones, cytokinins. The mutants have defects in cell division and elongation in the cotyledons and the apical root meristem. Cloning of pas-1 reveals that it is a homolog of mammalian FKBP52. FKBP52 has been shown to co-localize with the mitotic apparatus and to copurify with cytoplasmic dynein, suggesting that it too may be required for cell divisions (Munn, 2000 and references therein).

The shu phenotypes support the possibility that shu may also function during germline cell divisions. Specifically, its function seems to be important for the divisions of the germline stem cells. In support of this, expression of the protein can be detected in the stem cells while the levels are greatly reduced in the dividing cystocytes. The future identification of Shu-interacting proteins, coupled with the potential of genetic analysis in Drosophila, should greatly increase understanding of how germline stem cells are regulated and provide valuable information about the function of the immunophilins (Munn, 2000).


GENE STRUCTURE

cDNA clone length - 1682


PROTEIN STRUCTURE

Amino Acids - 392

Structural Domains

A search for recognized protein motifs, using the Prosite database, revealed a peptidyl-prolyl cis-trans isomerase (PPIASE) domain at amino acids 103-193 and one tetratricopeptide repeat (TPR) at amino acids 303-334. PPIASE domains are protein-protein interaction motifs that have been shown, in vitro, to catalyze the cis-trans isomerization of the peptide bond of proline residues, resulting in changes in protein folding (Fischer, 1990). Their activity is inhibited by binding of the drugs FK506 and rapamycin. Hence, proteins containing these domains are commonly referred to as FK506-binding proteins (FK506BP). The TPR motif is a degenerate 34-amino-acid sequence, which is also thought to mediate protein-protein interactions (Lamb, 1995). In addition, the Shu sequence contains a putative nuclear localization signal (NLS), and 18 predicted phosphorylation sites, although the functional significance of these, if any, remains unknown (Munn, 2000).

Comparison of the predicted amino acid sequence of Shu with other proteins in the database, using the Gapped BLAST search program, reveals that it is a novel Drosophila protein that shows similarity to proteins of the high-molecular-weight immunophilin family. This class of protein typically contains both PPIASE and TPR motifs. They are evolutionarily conserved, multifunctional proteins, examples of which have been identified in mammals and plants. Human FKBP6 (p36) (Meng, 1998) shows 28% identity and 46% similarity (probability 10-24 with 3% gaps) to Shu along the length of the entire protein sequence. A similar level of homology is also observed with human FKBP4 (Sanchez, 1990), FKBP52 (the mammalian p59 heat-shock protein-binding immunophilin; Peattie, 1992), and several plant immunophilins. Functional studies of human FKBP12, the best-characterized FKBP, have identified 14 residues important for enzymatic activity and drug binding (Kay, 1996). Shu retains 8 of these residues and a further amino acid is also conserved, suggesting that this domain is functionally important. Similarly, comparison of the Shu TPR motif with the consensus TPR sequence reveals that 5 of 6 consensus residues are conserved (Munn, 2000).


shutdown: Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

date revised: 25 May 2013

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