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. 2016 Nov;13(11):1117-1132.
doi: 10.1080/15476286.2016.1218592. Epub 2016 Aug 11.

The temporally controlled expression of Drongo, the fruit fly homolog of AGFG1, is achieved in female germline cells via P-bodies and its localization requires functional Rab11

Irina E Catrina  1 , Livia V Bayer  1   2 , Giussepe Yanez  1 , John M McLaughlin  1   2 , Kornelia Malaczek  1 , Ekaterina Bagaeva  1 , Salvatore A E Marras  3 , Diana P Bratu  1   2
Affiliations

The temporally controlled expression of Drongo, the fruit fly homolog of AGFG1, is achieved in female germline cells via P-bodies and its localization requires functional Rab11

Irina E Catrina et al. RNA Biol. 2016 Nov.

Abstract

To achieve proper RNA transport and localization, RNA viruses exploit cellular vesicular trafficking pathways. AGFG1, a host protein essential for HIV-1 and Influenza A replication, has been shown to mediate release of intron-containing viral RNAs from the perinuclear region. It is still unknown what its precise role in this release is, or whether AGFG1 also participates in cytoplasmic transport. We report for the first time the expression patterns during oogenesis for Drongo, the fruit fly homolog of AGFG1. We find that temporally controlled Drongo expression is achieved by translational repression of drongo mRNA within P-bodies. Here we show a first link between the recycling endosome pathway and Drongo, and find that proper Drongo localization at the oocyte's cortex during mid-oogenesis requires functional Rab11.

Keywords: AGFG1; Drongo; Drosophila; HIV-1; Influenza A; Rab11; cytoskeleton.

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Figures

Figure 1.
Figure 1.
Drosophila egg chamber and drongo gene model. (A) Model system used for these studies; stage 8 Drosophila egg chamber: oocyte, nurse cells, follicle cells, and ring canals (arrows). A = anterior, P = posterior. (B) RT-PCR results for full length drongo transcripts using total RNA isolated from wild type ovaries. Expected sizes for drongo-RI and drongo-RH transcripts are 2,625 and 3,518 nucleotides, respectively. (C) drongo gene model (FlyBase: Gbrowse): boxes highlight exons, orange for coding regions and gray for UTRs. On the 2L chromosome fragment (black ruler), black arrows indicate P-element insertion location in the drongo gene. Red arrows indicate the regions targeted by drongo-specific molecular beacons.
Figure 2.
Figure 2.
drongo mRNA localization in fixed and live oocytes using drongo-specific molecular beacons. (A) Detection of endogenous drongo transcript using dH1598-AF647 in fixed, wild type oocytes. drongo mRNA localization is indicated in the germline (arrows) and soma (asterisks). Microinjection of dH1598-AF647 in a nurse cell of a (B) wild type (XY-projection of 9 Z-slices/1 μm each) or (B’) GFP-Stau/CyO (XY-projection of 12 Z-slices/1 μm each) egg chamber at the indicated time points. Microinjection of dH1598-AF647 into a nurse cell of a (C) stage 7–8 (XY-projection of 5 Z-slices/1 μm each) or (C’) stage 10A (XY-projection of 5 Z-slices/1 μm each) Me31B-YFP egg chamber at the indicated time points. (D) Microinjection of dH1598-AF647 in a nurse cell of an armi mutant egg chamber (XY-projection of 10 Z-slices/1 μm each) at the indicated time points. s = stage. Bars, 20 μm.
Figure 3.
Figure 3.
Drongo overexpression during oogenesis. (A) Germline overexpression of Drongo-EGFP, and (A’) Clc-GFP. (B) Drongo-EGFP (MTD) localization at a nurse cell-oocyte ring canal. (B’) Normalized fluorescence intensity line plot profile along the line indicated in (B) for Drongo (green), F-actin (blue) and Hts (red). The GAL4 drivers used for Drongo-EGFP expression are indicated in parentheses. Arrows indicate (E)GFP accumulation in early stage oocytes. s = stage. Bars, 20 μm.
Figure 4.
Figure 4.
Drongo distribution in fixed egg chambers. (A) Detection of endogenous Drongo using immunofluorescence studies with Drongo peptide antibody in wild type egg chambers. (B) Endogenous Drongo localization in wild type and armi mutant oocytes. Arrow indicates ectopic Drongo accumulation. (C) Premature and ectopic accumulation of Drongo in stage<6 armi mutant oocytes resembles Drongo-EGFP clumps observed at a similar stage (arrows). (D) Premature expression of Drongo when Me31B expression is reduced using RNAi (MTD-GAL4; arrows; XY-projection of 5 Z-slices/0.5 μm each). F-actin was highlighted using fluorescently labeled phalloidin. s = stage. Bars, 20 μm.
Figure 5.
Figure 5.
drongo knockdown effects on egg morphology, and colocalization of Drongo with Clc. (A-B) Defects in drongoiHMJ as compared to (B’) wild type egg chamber. (A-A’) The oocyte position was highlighted with an oocyte marker, Bic-D (Bicaudal D). (B-B’) F-actin and Drongo distribution in one Z-slice, and rings canals in a XZ cross-section of drongoiHMJ (57 Z-slices/0.5 μm) or wild type (33 Z-slices/0.5 μm) egg chambers, respectively. (C) Comparison of AGFG1 and Drongo protein motifs (not drawn to scale). ZnF = C4H2 zinc finger, S/T = Serine/Threonine, FG = Phenylalanine and Glycine. Gray arrows indicate positions of the NPF (Asparagine-Proline-Phenylalanine) motifs. Arrow heads indicate the following: blue for AP2 (DLL, DxF or YxxΦ, Φ = hydrophobic residue), yellow for AP1 (DExxxL), and red for COPI (KKxx or WxxxW) binding motifs. (D) Colocalization between Drongo and Clc-GFP inside the oocyte (yellow: colocalization as XY-projection of 24 Z-slices/0.5 μm each, shown as an overlayer on the 9th Z-slice), and with F-actin at the anterior and posterior of a stage 8–9 oocyte. (D’) Localization of yolk granules and Drongo-EGFP particles in live egg chambers (>stage 9). (A-B’,D) F-actin was highlighted with fluorescently labeled phalloidin. A = anterior, P = posterior. Bars, (A-D) 20 μm and (D’) 10 μm.
Figure 6.
Figure 6.
Colocalization of Drongo with actin related proteins, or small GTPases at stages 8–9 of oogenesis. (A) Drongo-EGFP and Arpc3A in a stage 8–9 egg chamber. (B) Drongo-EGFP and Rab11 (yellow: colocalization as XY-projection of 61 Z-slices/0.5 μm each, shown as an overlayer on the 27th Z-slice), in a MTD>drongoEGFP stage 8–9 oocyte. F-actin was highlighted using fluorescently labeled phalloidin. (C) Drongo and Arf51F-GFP in a stage 8–9 egg chamber expressing endogenous GFP-tagged Arf51F under native promoter control. (A-C) Colocalization at the oocyte's cortex shown in a normalized fluorescence intensity profile measured along the lines drawn as shown in the merged panels. Arrows indicate colocalization at the cortex of the oocyte. A = anterior, P = posterior. Bars, 20 μm.
Figure 7.
Figure 7.
CK-666 treatment of live oocytes. F-actin and Drongo-EGFP localization for (A) control (DMSO) and (B) CK-666-treated stage 8 wild type and Drongo-EGFP oocytes. Arrows indicate ectopic F-actin clumps, and Drongo's localization at these sites. Bar, 10 μm.
Figure 8.
Figure 8.
Drongo and F-actin localization in YFP-Rab11 background. Expression of YFP-Rab11 and (A) Drongo or (B) Drongo and Drongo-EGFP, in the indicated background, at stages 8–9 of oogenesis. Bars, 20 μm.
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