Localization of the Drosophila Rad9 Protein to the Nuclear Membrane Is Regulated by the C-Terminal Region and Is Affected in the Meiotic Checkpoint

Rad9, Rad1, and Hus1 (9-1-1) are part of the DNA integrity checkpoint control system. It was shown previously that the C-terminal end of the human Rad9 protein, which contains a nuclear localization sequence (NLS) nearby, is critical for the nuclear transport of Rad1 and Hus1. In this study, we show that in Drosophila, Hus1 is found in the cytoplasm, Rad1 is found throughout the entire cell and that Rad9 (DmRad9) is a nuclear protein. More specifically, DmRad9 exists in two alternatively spliced forms, DmRad9A and DmRad9B, where DmRad9B is localized at the cell nucleus, and DmRad9A is found on the nuclear membrane both in Drosophila tissues and also when expressed in mammalian cells. Whereas both alternatively spliced forms of DmRad9 contain a common NLS near the C terminus, the 32 C-terminal residues of DmRad9A, specific to this alternative splice form, are required for targeting the protein to the nuclear membrane. We further show that activation of a meiotic checkpoint by a DNA repair gene defect but not defects in the anchoring of meiotic chromosomes to the oocyte nuclear envelope upon ectopic expression of non-phosphorylatable Barrier to Autointegration Factor (BAF) dramatically affects DmRad9A localization. Thus, by studying the localization pattern of DmRad9, our study reveals that the DmRad9A C-terminal region targets the protein to the nuclear membrane, where it might play a role in response to the activation of the meiotic checkpoint

Effects of meiotic checkpoint activation on DmRad9A oocyte nuclear membrane localization.

<p>Confocal images of stage 7 egg chambers. (A, E, I and M) are stained for DNA (blue, arrows mark oocyte nucleus DNA, karyosome); arrows mark the oocyte nucleus (karyosome). (B, F, J and N) GFP-DmRad9A (green). (C, G, K and O) are stained with anti-lamin antibodies, which mark the nuclear membrane, in red. (Inset in H, L and P), represents a schematic description of the oocyte nucleus. Red-lamin, green-GFP-DmRad9A and blue-karyosome. (A–H) GFP-DmRad9A:: <i>nosGal 4-VP16</i> egg chamber, E–H are enlargement of the oocyte region from A–D, respectively. (I-L) BAF3A:: GFP-DmRad9A:: <i>nosGal 4-VP16</i> egg chamber. M–P, GFP-DmRad9A:: <i>nosGal 4-VP16; okr^AA</i>/<i>okr^RU</i>.</p

Physical interaction between DmRad9, DmRad1 and DmHus1.

<p>DmRad9 was co-expressed in S2 cells with DmRad1 and DmHus1. A total lysate of S2 cells was extracted and subjected to immunoprecipitation. (A) DmRad1 was immunoprecipitated using anti-GFP antibodies. Anti-HA antibodies were used to detect DmHus1. (B) The same blot as in (A) was probed for FLAG-DmRad9 using anti-FLAG antibodies. (C–F) Confocal images of S₂R+ cells expressing FLAG-DmRad9, GFP-DmRad1 and HA-DmHus1. (G–J) Confocal images of follicle cells from transgenic FLAG-DmRad9::HA-DmHus1::GFP-DmRad1::<i>CY2Gal4</i> flies expressing egg chamber. (C) Staining with anti-FLAG antibodies detecting Flag-DmRad9. (D) Staining with anti-HA antibodies detecting HA-DmHus1. (E) GFP-DmRad1. (F) Merged (C–E). (G) Staining with anti-FLAG antibodies detecting Flag-DmRad9. (H) Staining with anti-HA antibodies detecting HA-DmHus1. (I) GFP-DmRad1. (J) merged G–I. Total protein served as positive control while a sample treated with protein A alone (no beads) served as negative control.</p

Localization of the <i>Drosophila</i> Rad9, Hus1 and Rad1 proteins in mammalian

<p>Human Embryonic Kidney 293 <b>(HEK293).</b> Confocal images of cells expressing (A) GFP-DmHus1, (D) GFP-DmRad1, and (G) GFP-DmRad9A. (B, E and H) Antibody staining of the NUP 414 protein, which recognizes several nucleoporins. (C, F and I) are merged images of (A–B), (D–E), and (G–H), respectively.</p

Identification of the DmRad9A nuclear localization signal.

<p>Confocal images of S₂R+ cells expressing DmRad9A mutated in suspected NLS sequences. (A) DmRad9A mutated at position 287 – 289 (NLS1). (D) DmRad9A mutated Position 300–302 (NLS2). (G) DmRad9A mutated Position 314–316 (NLS3). (B, E and H) stained with anti-lamin antibodies, which mark the nuclear membrane, in red. (C) Merged image of (A) and (B). (F) Merged image of (D) and (E). (I) Merged image of (G) and (H).</p

Localization of the <i>Drosophila</i> Rad9, Hus1 and Rad1 proteins in S₂R+ and follicle cells.

<p>A–G, Confocal images of S₂R+ cells, I–K, Confocal images of follicle cells from egg chambers. (A) S₂R+ cells expressi ng HA-DmHus1 and stained with anti-HA antibodies in red. (B) S₂R+ cells expressing GFP-DmRad1. (C) S₂R+ cells expressing GFP-DmRad9A. (F) S₂R+ cells expressing DmRad9B-GFP. (D) and (G) Staining with anti-lamin antibodies, which mark the nuclear membrane, in red. (E and H) are merged image of (C with differential interference contrast (DIC) image) and (F with a DIC image), respectively. (I) Egg chamber from HA-DmHus1::<i>CY2Gal4</i> transgenic flies. (J) Egg chamber from GFP-DmRad1::<i>CY2Gal4</i> transgenic flies. (K) Egg chamber from FLAG-DmRad9A::<i>CY2Gal4</i> transgenic flies. In both S₂R+ and follicle cells, DmHus1 is found in the cytoplasm, DmRad1 is found throughout the cell and Dm DmRad9A is localized to the nuclear membrane. DmRad9B is localized to the nucleus in S₂R+ cells.</p