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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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Targeted disruption of the mouse Rae1 gene. (A) Indicated are part of the endogenous mouse (m)Rae1 gene (top), the targeting vector (middle), and the disrupted Rae1 allele (bottom). BamHI restriction sites and the 3′ DNA probe (solid bar) used for Southern blot identification of wild-type (WT) and knockout (KO) Rae1 alleles are shown. (B) Southern blot of genomic ES cell DNA, digested with BamH1 and hybridized to a 3′ external probe, revealing the expected 10.2-kb wild-type and 8.5-kb mutant fragments. (C) PCR genotyping of E3.5 embryos from a Rae1+/− intercross showing wild-type and knockout allele-specific amplification products of respectively 516 and 650 bp. (D) In vitro growth of representative Rae1+/+ and Rae1−/− embryos. Embryos from heterozygous crosses were recovered at E3.5 and grown on multi-well slides in DME/15% FCS. They were inspected daily and photographed at 48 (E5.5), 72 (E6.5), and 96 h (E7.5) after seeding. I, ICM; T, trophectoderm cells.
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fig1: Targeted disruption of the mouse Rae1 gene. (A) Indicated are part of the endogenous mouse (m)Rae1 gene (top), the targeting vector (middle), and the disrupted Rae1 allele (bottom). BamHI restriction sites and the 3′ DNA probe (solid bar) used for Southern blot identification of wild-type (WT) and knockout (KO) Rae1 alleles are shown. (B) Southern blot of genomic ES cell DNA, digested with BamH1 and hybridized to a 3′ external probe, revealing the expected 10.2-kb wild-type and 8.5-kb mutant fragments. (C) PCR genotyping of E3.5 embryos from a Rae1+/− intercross showing wild-type and knockout allele-specific amplification products of respectively 516 and 650 bp. (D) In vitro growth of representative Rae1+/+ and Rae1−/− embryos. Embryos from heterozygous crosses were recovered at E3.5 and grown on multi-well slides in DME/15% FCS. They were inspected daily and photographed at 48 (E5.5), 72 (E6.5), and 96 h (E7.5) after seeding. I, ICM; T, trophectoderm cells.

Mentions: We addressed the physiological role of Rae1 through a gene-targeting approach in the mouse. We inactivated the Rae1 gene by interrupting its coding region at amino acid 50 with a lacZ-neo selection cassette (Fig. 1 A). Three independently targeted embryonic stem (ES) cell clones yielded chimeric mice that passed the disrupted Rae1 gene through the germline (Fig. 1 B). These Rae1 heterozygous mice were indistinguishable from wild-type littermates. We intercrossed heterozygous mice but found no Rae1−/− mice among 203 liveborn offspring, demonstrating that Rae1 is essential for mouse embryogenesis.


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)

Targeted disruption of the mouse Rae1 gene. (A) Indicated are part of the endogenous mouse (m)Rae1 gene (top), the targeting vector (middle), and the disrupted Rae1 allele (bottom). BamHI restriction sites and the 3′ DNA probe (solid bar) used for Southern blot identification of wild-type (WT) and knockout (KO) Rae1 alleles are shown. (B) Southern blot of genomic ES cell DNA, digested with BamH1 and hybridized to a 3′ external probe, revealing the expected 10.2-kb wild-type and 8.5-kb mutant fragments. (C) PCR genotyping of E3.5 embryos from a Rae1+/− intercross showing wild-type and knockout allele-specific amplification products of respectively 516 and 650 bp. (D) In vitro growth of representative Rae1+/+ and Rae1−/− embryos. Embryos from heterozygous crosses were recovered at E3.5 and grown on multi-well slides in DME/15% FCS. They were inspected daily and photographed at 48 (E5.5), 72 (E6.5), and 96 h (E7.5) after seeding. I, ICM; T, trophectoderm cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172680&req=5

fig1: Targeted disruption of the mouse Rae1 gene. (A) Indicated are part of the endogenous mouse (m)Rae1 gene (top), the targeting vector (middle), and the disrupted Rae1 allele (bottom). BamHI restriction sites and the 3′ DNA probe (solid bar) used for Southern blot identification of wild-type (WT) and knockout (KO) Rae1 alleles are shown. (B) Southern blot of genomic ES cell DNA, digested with BamH1 and hybridized to a 3′ external probe, revealing the expected 10.2-kb wild-type and 8.5-kb mutant fragments. (C) PCR genotyping of E3.5 embryos from a Rae1+/− intercross showing wild-type and knockout allele-specific amplification products of respectively 516 and 650 bp. (D) In vitro growth of representative Rae1+/+ and Rae1−/− embryos. Embryos from heterozygous crosses were recovered at E3.5 and grown on multi-well slides in DME/15% FCS. They were inspected daily and photographed at 48 (E5.5), 72 (E6.5), and 96 h (E7.5) after seeding. I, ICM; T, trophectoderm cells.
Mentions: We addressed the physiological role of Rae1 through a gene-targeting approach in the mouse. We inactivated the Rae1 gene by interrupting its coding region at amino acid 50 with a lacZ-neo selection cassette (Fig. 1 A). Three independently targeted embryonic stem (ES) cell clones yielded chimeric mice that passed the disrupted Rae1 gene through the germline (Fig. 1 B). These Rae1 heterozygous mice were indistinguishable from wild-type littermates. We intercrossed heterozygous mice but found no Rae1−/− mice among 203 liveborn offspring, demonstrating that Rae1 is essential for mouse embryogenesis.

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