The “Special” crystal-Stellate System in Drosophila melanogaster Reveals Mechanisms Underlying piRNA Pathway-Mediated Canalization

Hindawi Publishing Corporation Genetics Research International Volume 2012, Article ID 324293, 5 pages doi:10.1155/2012/324293 Review Article The “Special” crystal-Stellate System in Drosophila melanogaster Reveals Mechanisms Underlying piRNA Pathway-Mediated Canalization Maria Pia Bozzetti,1 Laura Fanti,2 Silvia Di Tommaso,1 Lucia Piacentini,2 Maria Berloco,3 Patrizia Tritto,3 and Valeria Specchia1 1 Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, 73100 Lecce, Italy 2 Sezione di Genetica, Dipartimento di Biologia e Biotecnologie “Charles Darwin”, Sapienza Università di Roma, 00185 Roma, Italy 3 Dipartimento di Biologia, Università degli Studi di Bari Aldo Moro, 70121 Bari, Italy Correspondence should be addressed to Maria Pia Bozzetti, Received 14 June 2011; Revised 18 August 2011; Accepted 21 September 2011 Academic Editor: Victoria H. Meller Copyright © 2012 Maria Pia Bozzetti et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Stellate-made crystals formation in spermatocytes is the phenotypic manifestation of a disrupted crystal-Stellate interaction in testes of Drosophila melanogaster. Stellate silencing is achieved by the piRNA pathway, but many features still remain unknown. Here we outline the important role of the crystal-Stellate modifiers. These have shed light on the piRNA pathways that defend genome integrity against transposons and other repetitive elements in the gonads. In particular, we illustrate the finding that HSP90 participates in the molecular pathways of piRNA production. This observation has relevance for the mechanisms underlying the evolutionary canalization process. 1. The Stellate-Made Crystals in Spermatocytes Are the Phenotypic Manifestation of a Disrupted crystal-Stellate Interaction in Testes of Drosophila melanogaster The history of the crystal-Stellate system started in 1961 when Meyer and collaborators discovered the presence of crystalline aggregates in primary spermatocytes of D. melanogaster X/O male testes. They also described the morphological differences between needle-shaped and star-shaped crystals [1]. In 1983, Gatti and Pimpinelli provided a detailed cytological description of the Y chromosome. They showed that the hll region contains the genetic determinants for normal chromosome behavior during male meiosis and for the suppression of Stellate-made crystals formation in spermatocytes [2]. This region was called the Suppressor of Stellate [Su(Ste)] locus, also referred to as crystal (cry) [3]; in this paper we use “crystal.” Afterwards, different groups established that both the morphology of the crystalline aggregates and the severity of the meiotic defects in X/O and X/Y cry- males depend on the Stellate (Ste) locus on the X chromosome [4–6]. Two regions containing clustered Stellate elements have been identified on the X chromosome: 12E1 in euchromatin and h27 in heterochromatin. Stellate and crystal are both repetitive sequences and they share sequence homology [6–8]. At the molecular level, the loss of the crystal region results in the production of a testes-specific Stellate mRNA of 750 nucleotides in length. The product of this mRNA is the Stellate protein [8, 9]. In 1995 there was a fundamental discovery: the Stellate protein is the main component of the crystals in the primary spermatocytes [10] and Figure 1. 2. The Regulation of the crystal-Stellate Interaction The first indication about the mechanism that regulates the interaction between crystal and Stellate sequences was 2 Genetics Research International (a) (b) Figure 1: Testes of X/Y cry- males immunostained with anti-Stellate antibody, (a) magnification 20x; (b) magnification 40x. obtained in 2001; the Stellate silencing was associated with the presence of small RNAs, 24–29 nt long, homologous to crystal and Stellate sequences [11]. These were named rasiRNAs (repeat-associated small interfering RNAs) [12]. The detailed analysis of the crystal-rasiRNAs in fly testes demonstrated the existence of a specific RNAi pathway in the germline that silences repetitive sequences such as Stellate and transposable elements [13]. It was also demonstrated that rasiRNAs show differences in structure compared to other classes of small noncoding RNAs, such as siRNAs and miRNAs and their biogenesis is Dicer-independent [13]. The rasiRNAs work associated with the Piwi subfamily of the Argonaute proteins, Aubergine, Ago3, and Piwi. rasiRNAs were subsequently designated as Piwi-interacting RNAs or piRNAs [13]. The studies on the crystal-Stellate system have been therefore crucial for the discovery of the piRNA pathway. In 2007, two independent groups used a deep sequencing strategy to identify small RNAs bound to each of the three Piwi proteins in fly ovaries. Their expectation was that this approach would reveal how piRNAs were made and how they function. They demonstrated that piRNAs arise from a few genomic sites, grouped in clusters that produce small RNAs that silence many transposons [14, 15]. In fly testes, the most abundant Aubergine-associated piRNAs (∼70%) correspond to crystal antisense transcripts [16]. 3. The piRNA Pathways in the Fly Ovaries Studies on the sequences of the small RNAs associated to Piwi subclade proteins carried out in 2006 and 2007 by the Hannon, Zamore, and Siomi groups have been crucial to formulation of a model for the biogenesis and the function of the piRNAs in the germline [13–16]. The proposed model, called the “ping-pong” model, requires a primary piRNA, whose biogenesis has not yet been elucidated, bound by Aubergine or Ago3. In particular, Aub binds an antisense piRNA and cleaves the sense transcript from an active transposon; transcript cleavage produces a sense piRNA that is loaded onto Ago3. This Ago3-piRNA complex binds complementary transcripts and initiates the production of piRNAs by an amplification loop [14]. The piRNAs originated by this mechanism are now called “secondary” piRNAs and they exhibit specific signatures consisting of the adenine at the 10th position of the sense piRNAs, which is able to base pair with the initial uracil of the antisense piRNAs [14, 15]. Identification of ago3 mutants led to the discovery of two different piRNA pathways in the fly ovary: one in the somatic cells of the ovary and the other in the germline cells. The somatic pathway, called “primary piRNA pathway,” involves Piwi, and it does not require an amplification loop. This pathway regulates the transposons belonging to the so-called “somatic” group [17, 18]. 4. The piRNA Pathways in Fly Testes and Open Questions Deep sequencing of piRNAs bound to Piwi-subfamily proteins associated to genetic studies, supplied thousands of data about almost all the piRNAs sequence biogenesis and orientation produced in testes [16, 19]. (...truncated)