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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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Disruption of a single Bub3 allele leads to mitotic checkpoint dysfunction. (A) Targeted inactivation of the mouse Bub3 gene. Shown is part of the endogenous mouse (m)Bub3 gene (top), the targeting vector (middle), and the disrupted mBub3 allele (bottom). BamHI restriction sites and the 5′ DNA probe (solid bar) used for Southern blot identification of wild-type (WT) and knockout (KO) Bub3 alleles are indicated. (B) Southern blot of genomic mouse tail DNA, digested with BamH1 and hybridized to a 5′ external probe, revealing the expected 7-kb wild-type and 5.5-kb mutant bands. (C) Western blot analysis of Bub3 protein levels in Bub3+/+ and Bub3+/− MEF lines using an antibody to mouse Bub3(145–276) (Wang et al., 2001). (D) Mitotic index of nocodazole-treated Bub3+/+ and Bub3+/− MEF cell lines. (E) Images of Bub3+/+ and Bub3+/− MEF cultures before and after nocodazole treatment. (F) DNA contents of asynchronous Bub3+/+ (panels 1–3) and Bub3+/− cells (panels 4–6) after the indicated treatments. (G) Cyclin B–associated Cdc2 kinase activity of synchronized MEF cultures at indicated time points after release into nocodazole.
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fig5: Disruption of a single Bub3 allele leads to mitotic checkpoint dysfunction. (A) Targeted inactivation of the mouse Bub3 gene. Shown is part of the endogenous mouse (m)Bub3 gene (top), the targeting vector (middle), and the disrupted mBub3 allele (bottom). BamHI restriction sites and the 5′ DNA probe (solid bar) used for Southern blot identification of wild-type (WT) and knockout (KO) Bub3 alleles are indicated. (B) Southern blot of genomic mouse tail DNA, digested with BamH1 and hybridized to a 5′ external probe, revealing the expected 7-kb wild-type and 5.5-kb mutant bands. (C) Western blot analysis of Bub3 protein levels in Bub3+/+ and Bub3+/− MEF lines using an antibody to mouse Bub3(145–276) (Wang et al., 2001). (D) Mitotic index of nocodazole-treated Bub3+/+ and Bub3+/− MEF cell lines. (E) Images of Bub3+/+ and Bub3+/− MEF cultures before and after nocodazole treatment. (F) DNA contents of asynchronous Bub3+/+ (panels 1–3) and Bub3+/− cells (panels 4–6) after the indicated treatments. (G) Cyclin B–associated Cdc2 kinase activity of synchronized MEF cultures at indicated time points after release into nocodazole.

Mentions: We then asked whether a haplo-insufficiency at the Bub3 locus would also cause a mitotic phenotype. We inactivated the mouse Bub3 gene by interrupting its coding region at amino acid 191 via homologous recombination (Fig. 5, A and B). Bub3+/− mice were indistinguishable from wild-type littermates (unpublished data). Like Rae1+/− intercrosses, Bub3+/− intercrosses yielded no Bub3−/− mice at birth and at day 8.5 of embryogenesis, but produced Bub3−/− blastocysts at a Mendelian frequency (unpublished data). The growth abnormalities of these Bub3-deficient blastocysts (unpublished data) were similar to those of Rae1−/− blastocysts (Fig. 1 D) and Bub3−/− blastocysts from an earlier study (Kalitsis et al., 2000). In situ hybridization experiments on E8.5 outgrowths confirmed that disruption of Bub3 had no impact on the localization and level of poly(A)+ RNA in the cell (unpublished data).


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)

Disruption of a single Bub3 allele leads to mitotic checkpoint dysfunction. (A) Targeted inactivation of the mouse Bub3 gene. Shown is part of the endogenous mouse (m)Bub3 gene (top), the targeting vector (middle), and the disrupted mBub3 allele (bottom). BamHI restriction sites and the 5′ DNA probe (solid bar) used for Southern blot identification of wild-type (WT) and knockout (KO) Bub3 alleles are indicated. (B) Southern blot of genomic mouse tail DNA, digested with BamH1 and hybridized to a 5′ external probe, revealing the expected 7-kb wild-type and 5.5-kb mutant bands. (C) Western blot analysis of Bub3 protein levels in Bub3+/+ and Bub3+/− MEF lines using an antibody to mouse Bub3(145–276) (Wang et al., 2001). (D) Mitotic index of nocodazole-treated Bub3+/+ and Bub3+/− MEF cell lines. (E) Images of Bub3+/+ and Bub3+/− MEF cultures before and after nocodazole treatment. (F) DNA contents of asynchronous Bub3+/+ (panels 1–3) and Bub3+/− cells (panels 4–6) after the indicated treatments. (G) Cyclin B–associated Cdc2 kinase activity of synchronized MEF cultures at indicated time points after release into nocodazole.
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Related In: Results  -  Collection

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fig5: Disruption of a single Bub3 allele leads to mitotic checkpoint dysfunction. (A) Targeted inactivation of the mouse Bub3 gene. Shown is part of the endogenous mouse (m)Bub3 gene (top), the targeting vector (middle), and the disrupted mBub3 allele (bottom). BamHI restriction sites and the 5′ DNA probe (solid bar) used for Southern blot identification of wild-type (WT) and knockout (KO) Bub3 alleles are indicated. (B) Southern blot of genomic mouse tail DNA, digested with BamH1 and hybridized to a 5′ external probe, revealing the expected 7-kb wild-type and 5.5-kb mutant bands. (C) Western blot analysis of Bub3 protein levels in Bub3+/+ and Bub3+/− MEF lines using an antibody to mouse Bub3(145–276) (Wang et al., 2001). (D) Mitotic index of nocodazole-treated Bub3+/+ and Bub3+/− MEF cell lines. (E) Images of Bub3+/+ and Bub3+/− MEF cultures before and after nocodazole treatment. (F) DNA contents of asynchronous Bub3+/+ (panels 1–3) and Bub3+/− cells (panels 4–6) after the indicated treatments. (G) Cyclin B–associated Cdc2 kinase activity of synchronized MEF cultures at indicated time points after release into nocodazole.
Mentions: We then asked whether a haplo-insufficiency at the Bub3 locus would also cause a mitotic phenotype. We inactivated the mouse Bub3 gene by interrupting its coding region at amino acid 191 via homologous recombination (Fig. 5, A and B). Bub3+/− mice were indistinguishable from wild-type littermates (unpublished data). Like Rae1+/− intercrosses, Bub3+/− intercrosses yielded no Bub3−/− mice at birth and at day 8.5 of embryogenesis, but produced Bub3−/− blastocysts at a Mendelian frequency (unpublished data). The growth abnormalities of these Bub3-deficient blastocysts (unpublished data) were similar to those of Rae1−/− blastocysts (Fig. 1 D) and Bub3−/− blastocysts from an earlier study (Kalitsis et al., 2000). In situ hybridization experiments on E8.5 outgrowths confirmed that disruption of Bub3 had no impact on the localization and level of poly(A)+ RNA in the cell (unpublished data).

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