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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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The mitotic checkpoint requires a full complement of Rae1. (A) Analysis of Rae1+/+ and Rae1+/− MEF lines for Rae1, Bub3, and Mad2 protein levels by Western blotting (4–20% polyacrylamide gel). 100 μg of total protein extract from each MEF cell line was used in the analysis. For probes, we used a rabbit antibody against mouse Rae1(188–368), a rabbit antibody against mouse Bub3(145–276), and a mouse monoclonal antibody against human Mad2 that recognizes mouse Mad2 (BD Biosciences). (B) Growth curves of primary MEFs. Data shown are means and standard deviations derived from three Rae1+/+ and three Rae1+/− MEF lines. (C) Mitotic index of nocodazole-treated Rae1+/− and Rae1+/+ MEF cell lines (n = 3 for each genotype). (D) Representative phase contrast images of Rae1+/+ and Rae1+/− MEF cultures before and after 4 h of nocodazole exposure. (E) Representative FACS® profiles of propidium-stained unsynchronized Rae1+/+ (panels 1–3) and Rae1+/− MEFs (panels 4–6). Durations of nocodazole treatment (80 ng/ml) and recovery intervals are indicated. (F) Cyclin B–associated Cdc2 kinase activity of synchronized MEF cells at indicated time points after release into nocodazole.
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fig3: The mitotic checkpoint requires a full complement of Rae1. (A) Analysis of Rae1+/+ and Rae1+/− MEF lines for Rae1, Bub3, and Mad2 protein levels by Western blotting (4–20% polyacrylamide gel). 100 μg of total protein extract from each MEF cell line was used in the analysis. For probes, we used a rabbit antibody against mouse Rae1(188–368), a rabbit antibody against mouse Bub3(145–276), and a mouse monoclonal antibody against human Mad2 that recognizes mouse Mad2 (BD Biosciences). (B) Growth curves of primary MEFs. Data shown are means and standard deviations derived from three Rae1+/+ and three Rae1+/− MEF lines. (C) Mitotic index of nocodazole-treated Rae1+/− and Rae1+/+ MEF cell lines (n = 3 for each genotype). (D) Representative phase contrast images of Rae1+/+ and Rae1+/− MEF cultures before and after 4 h of nocodazole exposure. (E) Representative FACS® profiles of propidium-stained unsynchronized Rae1+/+ (panels 1–3) and Rae1+/− MEFs (panels 4–6). Durations of nocodazole treatment (80 ng/ml) and recovery intervals are indicated. (F) Cyclin B–associated Cdc2 kinase activity of synchronized MEF cells at indicated time points after release into nocodazole.

Mentions: Western blot analysis revealed that the amount of Rae1 protein in Rae1+/− cells was consistently lower than in Rae1+/+ cell lines (Fig. 3 A); additional tests showed that Bub3 and Mad2 protein levels were similar in Rae1+/+ and Rae1+/− cells (Fig. 3 A). Growth rate measurements showed that the reduction of Rae1 protein had no significant impact on the rate of cell proliferation (Fig. 3 B). We then measured the response of the Rae1 haplo-insufficient cells to spindle damage. We treated cells with 200 ng/ml nocodazole for 0, 2, 6, 12, or 24 h, stained with phospho-histone H3 antibody to identify mitotic cells, and then measured the mitotic index (defined as the percentage of mitotic cells) at each time point using fluorescence microscopy. We found that 15% of Rae1+/+ cells were arrested by 12 h (Fig. 3 C). In contrast, only 2.5% of haplo-insufficient cells were arrested at that time point, indicating that their mitotic checkpoint was defective. Consistent with this observation, Rae1+/− cultures accumulated far fewer rounded cells in the presence of nocodazole than Rae1+/+ cultures (Fig. 3 D).


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)

The mitotic checkpoint requires a full complement of Rae1. (A) Analysis of Rae1+/+ and Rae1+/− MEF lines for Rae1, Bub3, and Mad2 protein levels by Western blotting (4–20% polyacrylamide gel). 100 μg of total protein extract from each MEF cell line was used in the analysis. For probes, we used a rabbit antibody against mouse Rae1(188–368), a rabbit antibody against mouse Bub3(145–276), and a mouse monoclonal antibody against human Mad2 that recognizes mouse Mad2 (BD Biosciences). (B) Growth curves of primary MEFs. Data shown are means and standard deviations derived from three Rae1+/+ and three Rae1+/− MEF lines. (C) Mitotic index of nocodazole-treated Rae1+/− and Rae1+/+ MEF cell lines (n = 3 for each genotype). (D) Representative phase contrast images of Rae1+/+ and Rae1+/− MEF cultures before and after 4 h of nocodazole exposure. (E) Representative FACS® profiles of propidium-stained unsynchronized Rae1+/+ (panels 1–3) and Rae1+/− MEFs (panels 4–6). Durations of nocodazole treatment (80 ng/ml) and recovery intervals are indicated. (F) Cyclin B–associated Cdc2 kinase activity of synchronized MEF cells at indicated time points after release into nocodazole.
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Related In: Results  -  Collection

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fig3: The mitotic checkpoint requires a full complement of Rae1. (A) Analysis of Rae1+/+ and Rae1+/− MEF lines for Rae1, Bub3, and Mad2 protein levels by Western blotting (4–20% polyacrylamide gel). 100 μg of total protein extract from each MEF cell line was used in the analysis. For probes, we used a rabbit antibody against mouse Rae1(188–368), a rabbit antibody against mouse Bub3(145–276), and a mouse monoclonal antibody against human Mad2 that recognizes mouse Mad2 (BD Biosciences). (B) Growth curves of primary MEFs. Data shown are means and standard deviations derived from three Rae1+/+ and three Rae1+/− MEF lines. (C) Mitotic index of nocodazole-treated Rae1+/− and Rae1+/+ MEF cell lines (n = 3 for each genotype). (D) Representative phase contrast images of Rae1+/+ and Rae1+/− MEF cultures before and after 4 h of nocodazole exposure. (E) Representative FACS® profiles of propidium-stained unsynchronized Rae1+/+ (panels 1–3) and Rae1+/− MEFs (panels 4–6). Durations of nocodazole treatment (80 ng/ml) and recovery intervals are indicated. (F) Cyclin B–associated Cdc2 kinase activity of synchronized MEF cells at indicated time points after release into nocodazole.
Mentions: Western blot analysis revealed that the amount of Rae1 protein in Rae1+/− cells was consistently lower than in Rae1+/+ cell lines (Fig. 3 A); additional tests showed that Bub3 and Mad2 protein levels were similar in Rae1+/+ and Rae1+/− cells (Fig. 3 A). Growth rate measurements showed that the reduction of Rae1 protein had no significant impact on the rate of cell proliferation (Fig. 3 B). We then measured the response of the Rae1 haplo-insufficient cells to spindle damage. We treated cells with 200 ng/ml nocodazole for 0, 2, 6, 12, or 24 h, stained with phospho-histone H3 antibody to identify mitotic cells, and then measured the mitotic index (defined as the percentage of mitotic cells) at each time point using fluorescence microscopy. We found that 15% of Rae1+/+ cells were arrested by 12 h (Fig. 3 C). In contrast, only 2.5% of haplo-insufficient cells were arrested at that time point, indicating that their mitotic checkpoint was defective. Consistent with this observation, Rae1+/− cultures accumulated far fewer rounded cells in the presence of nocodazole than Rae1+/+ cultures (Fig. 3 D).

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