Supplemental Material

This article contains the following supporting material:

• Figure 1 - (A, B) Preimmune sera for anti-dRrp6 and-dRrp40 antibodies used in Figure 1 detect non-specific signal. Images are 2-fold greater exposure time than images in Figure 1 to enhance visualization of background staining. Bar, 2 μm.

• Figure 2 - Cell Fractionation of Endogenous Exosome Subunits in Drosophila S2 Cells. (A) Endogenous dRrp6 is predominantly found in the presumptive nuclear fraction (pellet, P) whereas dRrp4, dRrp41, and dCsl4 are enriched in the soluble, presumptive cytoplasmic fraction (supernatant, S). The large subunit of RNA polymerase II shows equal distribution in both fractions. (B) Salt extraction profiles of exosome subunits during cell fractionation. To assess the solubility of endogenous exosome subunits under conditions used to make extracts for immunoaffinity purification experiments (Figure 3), we analyzed the cell fractionation profiles of endogenous polypeptides across a range of salt extraction conditions (0.005M to 0.6M NaCl). At the highest salt concentration, approximately 80% of endogenous dRrp6 is liberated from the insoluble fraction and > 90% of endogenous dRrp46, dRrp40, dRrp4, and dCsl4 are soluble (Figure 4B). In contrast, whereas Pol II fractionates equally in both soluble and insoluble fractions, the chromatin-associated transcription factor GAGA is liberated from chromatin between a salt range of 0.3M to 0.5M NaCl. Thus, we conclude that exosome subunits can be efficiently extracted from the nuclear pellet and from cytoplasmic strucutres.

• Figure 3 - RNAi depletion of dRrp4 does not affect dCsl4 stability. S2 cells treated with double-stranded RNA (dsRNA) to dRrp4 shown lower levels of the RNAi-targeted subunit. Panel represents a western blot of extracts from dsRNA-treated cells or and equivalent amount of untreated cells (serially diluted so as to determine depletion efficiency).

• Movie 1 - Volume projection of interphase S2 cell expressing dDis3FH and stained with anti-lamin (green), anti-FLAG (red), and DAPI (blue). Note the enrichment of dDis3FH on one side of the nuclear lamina.

• Movie 2 - Volume projection of interphase S2 cell expressing dRrp41FH and stained with anti-lamin (green), anti-FLAG (red), and DAPI (blue). dRrp41FH in this cell is predominantly nuclear. Note the enrichment of dRrp41FH on one side of the nuclear lamina.

• Movie 3 - Volume projection of interphase S2 cell expressing dRrp41FH and stained with anti-lamin (green), anti-FLAG (red), and DAPI (blue). dRrp41FH in this cell is predominantly cytoplasmic. dRrp41FH is enriched on one side of the nuclear lamina. Anti-lamin signal in lower left hand corner of video is from an adjacent cell. Very infrequently, dRrp41FH is found in cytoplasmic foci (Table 1); two of these foci are observed in this cell.

• Movie 4 - Volume projection of interphase S2 cell expressing dCsl4FH and stained with anti-lamin (green), anti-FLAG (red), and DAPI (blue). There are approximately 4 foci shown that are distal to the nucleus.

• Movie 5 - Volume projection of interphase S2 cell expressing dRrp42F and stained with anti-lamin (green), anti-FLAG (red), and DAPI (blue). This cell contains two small nuclear-proximal foci and three larger foci that appear to be contiguous with or near the cell membrane. Note that this three dimensional reconstruction allows for the detection of what appear to be nuclear foci.

• Movie 6 - Volume projection of interphase S2 cell expressing dRrp4FH and stained with anti-lamin (green), anti-FLAG (red), and DAPI (blue). In this cell, most of the cytoplasmic foci are adjacent to, or contiguous with, the nuclear rim. Note the variability in focus size.