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Rae1 is an essential mitotic checkpoint regulator that cooperates with Bub3 to prevent chromosome missegregation.

Babu JR, Jeganathan KB, Baker DJ, Wu X, Kang-Decker N, van Deursen JM - J. Cell Biol. (2003)

Bottom Line: Here we show that haplo-insufficiency of either Rae1 or Bub3 results in a similar phenotype involving mitotic checkpoint defects and chromosome missegregation.We also show that overexpression of Rae1 can correct for Rae1 haplo-insufficiency and, surprisingly, Bub3 haplo-insufficiency.Thus, our data demonstrate a novel function for Rae1 and characterize Rae1 and Bub3 as related proteins with essential, overlapping, and cooperating roles in the mitotic checkpoint.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, MN 55905, USA.

ABSTRACT
The WD-repeat proteins Rae1 and Bub3 show extensive sequence homology, indicative of functional similarity. However, previous studies have suggested that Rae1 is involved in the mRNA export pathway and Bub3 in the mitotic checkpoint. To determine the in vivo roles of Rae1 and Bub3 in mammals, we generated knockout mice that have these genes deleted individually or in combination. Here we show that haplo-insufficiency of either Rae1 or Bub3 results in a similar phenotype involving mitotic checkpoint defects and chromosome missegregation. We also show that overexpression of Rae1 can correct for Rae1 haplo-insufficiency and, surprisingly, Bub3 haplo-insufficiency. Rae1- and Bub3- mice are embryonic lethal, although cells from these mice did not have a detectable defect in nuclear export of mRNA. Unlike mice, compound haplo-insufficient Rae1/Bub3 mice are viable. However, cells from these mice exhibit much greater rates of premature sister chromatid separation and chromosome missegregation than single haplo-insufficient cells. Finally, we show that mice with mitotic checkpoint defects are more susceptible to dimethylbenzanthrene-induced tumorigenesis than wild-type mice. Thus, our data demonstrate a novel function for Rae1 and characterize Rae1 and Bub3 as related proteins with essential, overlapping, and cooperating roles in the mitotic checkpoint.

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Rae1 is not essential for nuclear export of mRNA. (A) Overview of the experimental design. Blastocysts from intercrosses of Rae1+/− mice were cultured for ∼4–5 d and then analyzed by immunostaining or in situ hybridization. (B–C') Double staining of E8.5 embryonic outgrowths with a polyclonal antibody against mouse Rae1(188–368) (Pritchard et al., 1999) and monoclonal antibody mAb414, a marker of the NPC (Wu et al., 2001). Shown are representative high-resolution images of trophectoderm cells. (D and E) Immunostaining of E8.5 embryonic outgrowths with a polyclonal antibody against Nup98(151–224) (Wu et al., 2001). Shown are high-resolution images of trophectoderm cells. (F and G) Localization of poly(A)+ RNA in trophectoderm cells from E8.5 Rae1+/+ and Rae1−/− outgrowths. A FITC–oligo(dT)50 probe was used for visualization of poly(A)+ by in situ hybridization. (H and I) Trophoblast cells stained with a polyclonal antibody against human Tap (Braun et al., 1999).
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fig2: Rae1 is not essential for nuclear export of mRNA. (A) Overview of the experimental design. Blastocysts from intercrosses of Rae1+/− mice were cultured for ∼4–5 d and then analyzed by immunostaining or in situ hybridization. (B–C') Double staining of E8.5 embryonic outgrowths with a polyclonal antibody against mouse Rae1(188–368) (Pritchard et al., 1999) and monoclonal antibody mAb414, a marker of the NPC (Wu et al., 2001). Shown are representative high-resolution images of trophectoderm cells. (D and E) Immunostaining of E8.5 embryonic outgrowths with a polyclonal antibody against Nup98(151–224) (Wu et al., 2001). Shown are high-resolution images of trophectoderm cells. (F and G) Localization of poly(A)+ RNA in trophectoderm cells from E8.5 Rae1+/+ and Rae1−/− outgrowths. A FITC–oligo(dT)50 probe was used for visualization of poly(A)+ by in situ hybridization. (H and I) Trophoblast cells stained with a polyclonal antibody against human Tap (Braun et al., 1999).

Mentions: The ability to culture Rae1−/− blastocysts allowed us to further investigate the role of Rae1 in nucleocytoplasmic transport (Fig. 2 A). First, we double stained E7.5–E8.5 embryonic outgrowths with a polyclonal antibody against mouse Rae1(188–368) and monoclonal antibody mAb414, a marker of the nuclear pore complex (NPC) (Wu et al., 2001). In cells from control embryonic outgrowths, Rae1 prominently localized to the nuclear envelope (NE), although significant amounts of Rae1 were also found in the nucleus and the cytoplasm (Fig. 2 B). No Rae1 staining was detected in cells from Rae1−/− outgrowths, confirming that our gene-targeting strategy had indeed generated a allele (Fig. 2 C). Disruption of the Rae1 homologue GLE2 in Saccharomyces cerevisiae causes severe clustering of nuclear pores (Murphy and Wente, 1996). Rae1-depleted cells showed a strong and uninterrupted rim-like labeling of the NE with mAb414, similar to that of control cells (Fig. 2 C′), indicating that knockout cells have a normal distribution of NPCs. Another feature of Gle2p-deficient yeast cells is the formation of membranous structures that seal nuclear pores (Murphy and Wente, 1996). Examination of embryonic outgrowths by transmission electron microscopy demonstrated that NPC sealing does not occur in Rae1−/− cells (unpublished data). Nup98, a nucleoporin that forms a complex with Rae1 at the NPC (Pritchard et al., 1999), exhibited a pronounced NE localization in Rae1−/− embryos (Fig. 2, D and E), demonstrating that Rae1 is not needed for binding of Nup98 to NPCs.


Rae1 is an essential mitotic checkpoint regulator that cooperates with Bub3 to prevent chromosome missegregation.

Babu JR, Jeganathan KB, Baker DJ, Wu X, Kang-Decker N, van Deursen JM - J. Cell Biol. (2003)

Rae1 is not essential for nuclear export of mRNA. (A) Overview of the experimental design. Blastocysts from intercrosses of Rae1+/− mice were cultured for ∼4–5 d and then analyzed by immunostaining or in situ hybridization. (B–C') Double staining of E8.5 embryonic outgrowths with a polyclonal antibody against mouse Rae1(188–368) (Pritchard et al., 1999) and monoclonal antibody mAb414, a marker of the NPC (Wu et al., 2001). Shown are representative high-resolution images of trophectoderm cells. (D and E) Immunostaining of E8.5 embryonic outgrowths with a polyclonal antibody against Nup98(151–224) (Wu et al., 2001). Shown are high-resolution images of trophectoderm cells. (F and G) Localization of poly(A)+ RNA in trophectoderm cells from E8.5 Rae1+/+ and Rae1−/− outgrowths. A FITC–oligo(dT)50 probe was used for visualization of poly(A)+ by in situ hybridization. (H and I) Trophoblast cells stained with a polyclonal antibody against human Tap (Braun et al., 1999).
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fig2: Rae1 is not essential for nuclear export of mRNA. (A) Overview of the experimental design. Blastocysts from intercrosses of Rae1+/− mice were cultured for ∼4–5 d and then analyzed by immunostaining or in situ hybridization. (B–C') Double staining of E8.5 embryonic outgrowths with a polyclonal antibody against mouse Rae1(188–368) (Pritchard et al., 1999) and monoclonal antibody mAb414, a marker of the NPC (Wu et al., 2001). Shown are representative high-resolution images of trophectoderm cells. (D and E) Immunostaining of E8.5 embryonic outgrowths with a polyclonal antibody against Nup98(151–224) (Wu et al., 2001). Shown are high-resolution images of trophectoderm cells. (F and G) Localization of poly(A)+ RNA in trophectoderm cells from E8.5 Rae1+/+ and Rae1−/− outgrowths. A FITC–oligo(dT)50 probe was used for visualization of poly(A)+ by in situ hybridization. (H and I) Trophoblast cells stained with a polyclonal antibody against human Tap (Braun et al., 1999).
Mentions: The ability to culture Rae1−/− blastocysts allowed us to further investigate the role of Rae1 in nucleocytoplasmic transport (Fig. 2 A). First, we double stained E7.5–E8.5 embryonic outgrowths with a polyclonal antibody against mouse Rae1(188–368) and monoclonal antibody mAb414, a marker of the nuclear pore complex (NPC) (Wu et al., 2001). In cells from control embryonic outgrowths, Rae1 prominently localized to the nuclear envelope (NE), although significant amounts of Rae1 were also found in the nucleus and the cytoplasm (Fig. 2 B). No Rae1 staining was detected in cells from Rae1−/− outgrowths, confirming that our gene-targeting strategy had indeed generated a allele (Fig. 2 C). Disruption of the Rae1 homologue GLE2 in Saccharomyces cerevisiae causes severe clustering of nuclear pores (Murphy and Wente, 1996). Rae1-depleted cells showed a strong and uninterrupted rim-like labeling of the NE with mAb414, similar to that of control cells (Fig. 2 C′), indicating that knockout cells have a normal distribution of NPCs. Another feature of Gle2p-deficient yeast cells is the formation of membranous structures that seal nuclear pores (Murphy and Wente, 1996). Examination of embryonic outgrowths by transmission electron microscopy demonstrated that NPC sealing does not occur in Rae1−/− cells (unpublished data). Nup98, a nucleoporin that forms a complex with Rae1 at the NPC (Pritchard et al., 1999), exhibited a pronounced NE localization in Rae1−/− embryos (Fig. 2, D and E), demonstrating that Rae1 is not needed for binding of Nup98 to NPCs.

Bottom Line: Here we show that haplo-insufficiency of either Rae1 or Bub3 results in a similar phenotype involving mitotic checkpoint defects and chromosome missegregation.We also show that overexpression of Rae1 can correct for Rae1 haplo-insufficiency and, surprisingly, Bub3 haplo-insufficiency.Thus, our data demonstrate a novel function for Rae1 and characterize Rae1 and Bub3 as related proteins with essential, overlapping, and cooperating roles in the mitotic checkpoint.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, MN 55905, USA.

ABSTRACT
The WD-repeat proteins Rae1 and Bub3 show extensive sequence homology, indicative of functional similarity. However, previous studies have suggested that Rae1 is involved in the mRNA export pathway and Bub3 in the mitotic checkpoint. To determine the in vivo roles of Rae1 and Bub3 in mammals, we generated knockout mice that have these genes deleted individually or in combination. Here we show that haplo-insufficiency of either Rae1 or Bub3 results in a similar phenotype involving mitotic checkpoint defects and chromosome missegregation. We also show that overexpression of Rae1 can correct for Rae1 haplo-insufficiency and, surprisingly, Bub3 haplo-insufficiency. Rae1- and Bub3- mice are embryonic lethal, although cells from these mice did not have a detectable defect in nuclear export of mRNA. Unlike mice, compound haplo-insufficient Rae1/Bub3 mice are viable. However, cells from these mice exhibit much greater rates of premature sister chromatid separation and chromosome missegregation than single haplo-insufficient cells. Finally, we show that mice with mitotic checkpoint defects are more susceptible to dimethylbenzanthrene-induced tumorigenesis than wild-type mice. Thus, our data demonstrate a novel function for Rae1 and characterize Rae1 and Bub3 as related proteins with essential, overlapping, and cooperating roles in the mitotic checkpoint.

Show MeSH
Related in: MedlinePlus