(A) Piwi expression is detected in Drosophila ovaries and embryos, but not in S2 cells. Western blotting was performed on lysates of fly embryo (at early and late stages), ovary, and S2 cells using an antibody against Piwi. Anti-tubulin was utilized as a loading control. (B) Piwi expression pattern in ovary and testis. All the images shown in this figure represent one confocal section. (Panels II,III,IV) The images of ovariole staining show that Piwi (red) is found clearly to be accumulated in nuclei of both GSCs and somatic cells such as TFCs, cap cells (CpC), and follicle cells (FC). Panel I shows the DIC image of panel II. (Panel V–VIII) In testis, Piwi (red) is expressed in the hub (shown in green; stained with anti-DE-cadherin) at the apical tip of the testis and in the cyst progenitor cells (CC). Male GSCs are not stained with anti-Piwi. Panel VI shows the enlarged image of a part of panel V. Panel VIII shows a merged image of panels VI and VII.

(A) Protein components of the Piwi complex immunopurified from ovary lysate are visualized by silver staining. A strong discrete band appearing at ∼92 kDa on the gel is Piwi. h.c. shows where the heavy chain of the antibodies used migrated on the gel, and n.i. indicates nonimmune antibody used as a negative control. (B) Western blotting with anti-Piwi on the immunoprecipitates shown in A confirms that the 92-kDa band is indeed Piwi. (C) Visualization of small RNAs associated with Piwi in ovary. RNA molecules were isolated from the Piwi immunopurified complex shown in A, labeled with ³²P-pCp, and run on a 7 M urea-containing denaturing acrylamide gel. RNAs associated with AGO1 in ovary involve molecules ∼21–23 nt in length (blue lines), which from their size are expected to be miRNAs. Piwi-associated small RNAs (red lines) migrated slightly slower than miRNAs. The size was estimated at ∼25–29 nt in length. (n.i.) Nonimmune antibody used as a negative control. (D) Northern blot analyses using specific probes for miR-1 and miR-310 show that Piwi-associated small RNAs do not include the miRNAs.

Identification of small RNAs associated with Piwi in ovary. (A) The regions where roo, 412, and stalker4 rasiRNAs are identified as Piwi-associated rasiRNAs corresponding to the transcripts are shown by arrowheads. rasiRNAs in sense and antisense orientations are indicated in white and black, respectively. (B) The first nucleotide of the rasiRNA associated with Piwi is predominantly U (1st). This tendency was not found to be obvious in the second and last nucleotides of the rasiRNAs.

Specificities of the Piwi–rasiRNA association. (A) RNAs associated with Piwi in ovary were isolated and probed with sense and antisense roo transcripts internally labeled with ³²P in vitro. The left panel shows that Piwi associates with roo anti-rasiRNAs originating from roo antisense transcript. The right panel shows the Piwi association with roo rasiRNAs that is derived from the roo transcript in sense orientation. roo sense and antisense DNA oligos (10 fmol each) were also loaded on gels to show the probe specificities. roo anti-rasiRNA signal in the anti-Piwi lane was several-fold stronger than that of roo antisense oligo (left panel), whereas roo rasiRNA signal in the anti-Piwi lane (right) is a little less (∼85%) compared with that of the roo sense oligo, indicating that Piwi likely associates preferentially with roo anti-rasiRNA. Note that the AGO1 immunopurified complex (both panels) does not contain roo rasiRNAs, showing the specificity of the Piwi–rasiRNA association. (B) Blots containing RNAs associated with Piwi and AGO1 in ovary were probed with copia and TART transcripts and one of repetitive elements, Responder. Small RNAs corresponding to copia and TART rasiRNAs are detected only in the anti-Piwi lanes, indicating that the association with both rasiRNAs is specific for Piwi. (C) siRNA preprogrammed in ovary lysate was loaded onto AGO2, but not onto Piwi. Ovary lysate was prepared first and luc siRNA duplex was added to the lysate. After incubation, AGO2 and Piwi were immunopurified from the mixture using the specific antibodies. RNA molecules were then isolated from the complexes and probed with luc siRNA passenger strand to visualize the luc guide siRNA. In the anti-AGO2 lane, the luc guide siRNA strongly appeared, but not in the anti-Piwi and n.i. (nonimmune) lanes, indicating that luc guide siRNA was specifically loaded onto AGO2.

Target RNA cleavage assay with GST-Piwi and GSTAGO1. Recombinant full-length Piwi and AGO1 fused to GST were produced in and purified from Escherichia coli. GST was used as a control. As guide siRNA, luc 21 (luc guide siRNA, 21 nt; Miyoshi et al. 2005) and bantam miRNA (21 nt; Miyoshi et al. 2005) were used. GST fusion proteins were first incubated with guide siRNA, and then the luc target labeled at the 5′ end (Miyoshi et al. 2005) was added to the reaction mixture. After incubation, resultant RNAs were loaded on a denaturing acrylamide gel. The cleaved product was observed not only with GST-AGO1, but also with GST-Piwi. GST-AGO1 bound with bantam did not cleave the target, as expected.