PIWI-interacting RNAs (piRNAs) silence retrotransposons in Drosophila germ lines by associating with the PIWI proteins Argonaute 3 (AGO3), Aubergine (Aub) and Piwi. piRNAs in Drosophila are produced from intergenic repetitive genes and piRNA clusters by two systems: the primary processing pathway and the amplification loop. The amplification loop occurs in a Dicer-independent, PIWI-Slicer-dependent manner. However, primary piRNA processing remains elusive. Here we analysed piRNA processing in a Drosophila ovarian somatic cell line where Piwi, but not Aub or AGO3, is expressed; thus, only the primary piRNAs exist. In addition to flamenco, a Piwi-specific piRNA cluster, traffic jam (tj), a large Maf gene, was determined as a new piRNA cluster. piRNAs arising from tj correspond to the untranslated regions of tj messenger RNA and are sense-oriented. piRNA loading on to Piwi may occur in the cytoplasm. zucchini, a gene encoding a putative cytoplasmic nuclease, is required for tj-derived piRNA production. In tj and piwi mutant ovaries, somatic cells fail to intermingle with germ cells and Fasciclin III is overexpressed. Loss of tj abolishes Piwi expression in gonadal somatic cells. Thus, in gonadal somatic cells, tj gives rise simultaneously to two different molecules: the TJ protein, which activates Piwi expression, and piRNAs, which define the Piwi targets for silencing.

Transcriptome analyses in eukaryotes, including mice and humans, have identified polyA-containing transcripts that lack long open reading frames (ORFs; >100 amino acids). These transcripts are believed most likely to function as non-coding RNAs, but their translational capacities and biological activities have not been characterized in detail. Here, we report that polished rice (pri), which was previously identified as a gene for a non-coding RNA in Drosophila, is in fact transcribed into a polycistronic mRNA that contains evolutionarily conserved short ORFs that encode 11 or 32 amino acid-long peptides. pri was expressed in all epithelial tissues during embryogenesis. The loss of pri function completely eliminated apical cuticular structures, including the epidermal denticles and tracheal taenidia, and also caused defective tracheal-tube expansion. We found that pri is essential for the formation of specific F-actin bundles that prefigures the formation of the denticles and taenidium. We provide evidences that pri acts non-cell autonomously and that four of the conserved pri ORFs are functionally redundant. These results demonstrate that pri has essential roles in epithelial morphogenesis by regulating F-actin organization.

Genetic studies have shown that Aubergine (Aub), one of the Piwi subfamily of Argonautes in Drosophila, is essential for germ cell formation and maintaining fertility. aub mutations lead to the accumulation of retrotransposons in ovaries and testes, and Stellate transcripts in testes. Aub in ovaries associates with a variety of Piwi-interacting RNAs (piRNAs) derived from repetitive intergenic elements including retrotransposons. Here we found that Aub in testes also associates with various kinds of piRNAs. Although in ovaries Aub-associated piRNA populations are quite diverse, piRNAs with Aub in testes show a strong bias. The most abundant piRNAs were those corresponding to antisense transcripts of Suppressor of Stellate [Su(Ste)] genes known to be involved in Stellate gene silencing. The second most abundant class was made up of those from chromosome X and showed strong complementarity to vasa transcripts. Immunopurified Aub-piRNA complexes from testes displayed activity in cleaving target RNA containing sequences complementary to Stellate and vasa transcripts. These results provide the first biochemical insights into gene silencing mechanisms mediated by Aub and piRNAs in fly testes.

In Drosophila, the PIWI proteins, Aubergine (Aub), AGO3, and Piwi are expressed in germlines and function in silencing transposons by associating with PIWI-interacting RNAs (piRNAs). Recent studies show that PIWI proteins contain symmetric dimethyl-arginines (sDMAs) and that dPRMT5/Capsuleen/DART5 is the modifying enzyme. Here, we show that Tudor (Tud), one of Tud domaincontaining proteins, associates with Aub and AGO3, specifically through their sDMA modifications and that these three proteins form heteromeric complexes. piRNA precursor-like molecules are detected in these complexes. The expression levels of Aub and AGO3, along with their degree of sDMA modification, were not changed by tud mutations. However, the population of transposon-derived piRNAs associated with Aub and AGO3 was altered by tud mutations, whereas the total amounts of small RNAs on Aub and AGO3 was increased. Loss of dprmt5 did not change the stability of Aub, but impaired its association with Tud and lowered piRNA association with Aub. Thus, in germline cells, piRNAs are quality-controlled by dPRMT5 that modifies PIWI proteins, in tight association with Tud.