InteractiveFly: GeneBrief

vreteno: Biological Overview | References

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. This study 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. 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. It is proposed that Vret plays an essential role in transposon regulation at an early stage of primary piRNA processing (Zamparini, 2011).

Propagation of all sexually reproducing organisms depends upon the faithful development and function of reproductive organs. In Drosophila, oogenesis requires the coordinated differentiation of two distinct cell lineages, the germline and the gonadal somatic cells, to produce an egg. The germarium, where oogenesis initiates, contains both germline and somatic stem cells. Asymmetric cell division of germline stem cells (GSCs) within the germarium generates both a stem cell and a differentiated daughter cell, the cystoblast, which gives rise to a sixteen-cell interconnected cyst. One of the sixteen cells in the cyst differentiates into an egg and the remaining cells become nurse cells. Somatic cell populations are intimately associated with germ cells during oogenesis: niche cells provide GSC maintenance signals and are tightly connected to GSCs via adhesion and gap junctions; inner sheath cells (ISCs) intermingle with the differentiating cystoblast and early dividing cysts to promote formation of the sixteen-cell cyst; follicle stem cells and their progeny, the follicle cells, surround each germline cyst as it buds off from the germarium and provide the maturing egg chamber with the positional cues needed for establishment of anterior-posterior and dorsal-ventral polarity of the embryo (Zamparini, 2011).

In addition to germline development, genomic integrity must be preserved to generate viable progeny. In Drosophila, transposable elements occupy nearly one third of the genome and mobilization of even one of almost 150 transposon classes found can lead to defects in gametogenesis and sterility. Therefore, organisms have evolved small RNA-based defense systems to fight these elements (Malone, 2009). In Drosophila, both germline and somatic cells of the ovary rely on Piwi proteins and their 23-29 nt Piwi-interacting RNAs (piRNAs) to combat transposon activity. All three Drosophila Piwi proteins, Piwi, Aubergine (Aub) and Argonaute 3 (AGO3), are expressed in germline cells, whereas Piwi is also expressed in somatic gonadal cells. Interestingly, mutations in all known piRNA pathway components lead to oocyte and embryonic patterning defects and, ultimately, to sterility, believed to be an indirect consequence of transposon-induced genomic instability and activation of a DNA double-strand break checkpoint (Zamparini, 2011 and references therein).

In contrast to other small RNAs, such as microRNAs and siRNAs, which are produced from double-stranded RNA precursors, piRNAs are derived from single-stranded RNA precursors, independently of the endonuclease Dicer. piRNA precursors originate from either active transposon transcripts or discrete genomic loci known as 'piRNA clusters'. In Drosophila, piRNA clusters provide the primary source of antisense transposon transcripts, whereas active transposons predominantly provide sense transcripts. piRNAs associated with Piwi and Aub are mostly derived from piRNA clusters, mapping complementary to active transposons, whereas AGO3-bound piRNAs appear to be derived from the transposon itself. This relationship and a 10 nt overlap observed between sense and antisense piRNA pairs led to a model of piRNA amplification termed 'ping-pong', in which 5' ends of new piRNAs are generated through cleavage by the Piwi proteins themselves (Brennecke, 2007; Gunawardane, 2007). In the Drosophila ovary, piRNA `ping-pong' is restricted to germline cells in which Piwi, Aub and AGO3 are present, although Piwi appears to be mostly dispensable for 'ping-pong' amplification (Malone, 2009). In gonadal somatic cells, in which only Piwi is expressed, an alternative pathway functions. Here, single-stranded piRNA clusters or gene transcripts are processed to produce 'primary' piRNAs that are directly loaded into Piwi, targeting active transposons or endogenous genes (Li, 2009; Malone, 2009; Saito, 2009). The overlapping genetic requirements for Piwi in the germline and ovarian somatic cells suggest that Piwi may also engage primary piRNAs in the germline. Like Piwi, the germline-specific Aub engages piRNAs complementary to transposons, but has not been directly linked to primary piRNAs. Therefore, the precise relationship between primary piRNAs and 'ping-pong' in the germline remains largely unknown (Zamparini, 2011).

The restriction of piRNA production and transposon control in gonadal tissues raises the question of how the piRNA biogenesis machinery has evolved specifically in the gonad. This study has identified Vreteno (Vret), a gonad-specific, Tudor domain-containing protein that functions specifically in the germline and somatic gonadal tissues during oogenesis. Vret broadly regulates transposon levels and has an essential role in primary piRNA biogenesis, leaving 'ping-pong' amplification intact (Zamparini, 2011).

This study identified a novel protein with critical roles in oocyte polarity, germline and soma differentiation, survival and transposon control. Vret, a Tudor-domain containing protein, associates with Piwi proteins in the cytoplasm of Drosophila ovarian cells and regulates their stability, as well as Piwi nuclear localization and localization of Aub to nuage. In the absence of Vret, piRNAs are dramatically reduced and transposons mobilized. By ordering the function of Vret within the network of the piRNA-transposon-based system, it is concluded that Vret functions in primary piRNA biogenesis at the stage of primary piRNA loading onto Piwi and Aub complexes (Zamparini, 2011).

Loss of Vret in the soma or germline has strikingly different morphological consequences. Molecular analysis, however, suggests the same underlying cause for these defects: a failure to produce biologically active piRNAs. Morphologically, the vret germline phenotype resembles that of mutants defective in germline piRNA biogenesis, such as aub, spnE and krimper. In these mutants, transposon mobilization activates a DNA damage checkpoint that leads to defects in transport and translation of maternal RNAs necessary for oocyte polarity and embryonic patterning. Interestingly, lack of vret in the soma resembles the piwi mutant phenotype, in which GSCs fail to differentiate as a consequence of somatic cell death, an event presumably associated with transposon misregulation. Thus, loss of vret in the germline and gonadal soma resembles loss of both Piwi and Aub. This, together with the findings that Vret associates with Piwi and Aub in ovarian extracts and affects the stability of both, strongly suggests that Vret regulates both proteins in a similar fashion (Zamparini, 2011).

Surprisingly, Vret is not required for piRNA 'ping-pong' amplification per se, suggesting that Vret might selectively interact with Aub and Piwi bound to primary piRNAs and not to those engaged in 'ping-pong'. In this scenario, it would be possible for maternally deposited Aub to initiate the 'ping-pong' cycle with AGO3, even in the absence of Vret (Brennecke, 2008). As some Aub protein remains in vret mutant ovaries, an active pool of Vret-independent Aub could maintain 'ping-pong' activity throughout the adult ovary. Therefore, it is proposed that a 'ping-pong'-independent pool of Aub within the cytoplasm depends upon primary piRNA loading, downstream of Vret function. It would be interesting to examine whether piRNAs associated with the Vret-dependent complex can, at any level, contribute to 'ping-pong', or whether Aub-bound primary piRNAs are functionally or enzymatically distinct from those involved in the piRNA amplification cycle (Zamparini, 2011).

In contrast to Aub, only a small subset of Piwi-bound piRNAs showed a 10 nt overlap with those bound to AGO3. Indeed, Piwi is genetically dispensible for 'ping-pong' and might be only marginally involved in 'ping-pong', if at all (Brennecke, 2007; Li, 2009). As Piwi slicer activity does not appear to be required for Piwi function (Saito, 2009), it seems most plausible that Piwi would act as a recipient, and not as an 'active' component of 'ping-pong' amplification. Regardless, the majority of Piwi-bound primary piRNAs act independently of 'ping-pong' and depend upon Vret for stability (Zamparini, 2011).

An ectopic expression experiment suggests that Piwi is not 'intrinsically unstable', but becomes unstable in the gonad in the absence of Vret. Furthermore, Vret is not required for Piwi or Aub transcription or translation. Vret, therefore, could either coordinate the process of biogenesis and loading of primary piRNAs into Piwi and Aub complexes or be involved in stabilizing the mature RISC (RNA-induced silencing complex). Armi, a putative helicase, and Zucchini (Zuc), a member of the phospholipase D (PLD) family of phosphodiesterases, act like Vret in the soma and germline; they specifically affect Piwi protein stability and primary piRNA levels leaving the 'ping-pong' cycle intact. Unlike Vret, the levels of unprocessed precursor RNA from flam are increased in zuc mutants implicating Zuc in piRNA cluster transcript processing. Therefore the hypothesis is favored that Vret, possibly together with Armi, is an essential component of Piwi and Aub RISC complexes. Vret is one of many Tudor domain proteins in Drosophila that affects piRNA biogenesis and contains conserved residues that are known to be required for binding of sDMAs found in Piwi proteins (Siomi, 2010). When mutated, each of these genes displays a rather distinct phenotype. Krimper and SpnE regulate transposon levels in the germline whereas fs(1)Yb is soma-specific. Vret is, at this point, the only Tudor domain protein known to be required in both tissues, suggesting a conserved and global role for this gene in piRNA regulation. It remains to be determined whether the mammalian Tudor homolog could fulfill a similar function (Zamparini, 2011).

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Search PubMed for articles about Drosophila Vreteno

Brennecke, J., et al. (2007). Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128: 1089-1103. PubMed ID: 17346786

Gunawardane. L. S., et al. (2007). A slicer-mediated mechanism for repeat-associated siRNA 5' end formation in Drosophila. Science 315: 1587-1590. PubMed ID: 17322028

Li, C., et al. (2009). Collapse of germline piRNAs in the absence of Argonaute3 reveals somatic piRNAs in flies. Cell 137: 509-521. PubMed ID: 19395009

Malone, C. D., et al. (2009). Specialized piRNA pathways act in germline and somatic tissues of the Drosophila ovary. Cell 137: 522-535. PubMed ID: 19395010

Saito, K., et al. (2009). A regulatory circuit for piwi by the large Maf gene traffic jam in Drosophila. Nature 461: 1296-1299. PubMed ID: 19812547

Siomi, M. C., Mannen, T. and Siomi H. (2010). How does the royal family of Tudor rule the PIWI-interacting RNA pathway? Genes Dev. 24: 636-646. PubMed ID: 20360382

Zamparini, A. L., et al. (2011). Vreteno, a gonad-specific protein, is essential for germline development and primary piRNA biogenesis in Drosophila. Development 138(18): 4039-50. PubMed ID: 21831924

date revised: 25 April 2012

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