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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 synergizes with Bub3 in the development of aneuploidy. (A) Growth curves of MEFs derived from wild-type, Rae1+/−, Bub3+/−, and Rae1+/−/Bub3+/− 13.5-d-old embryos (all harvested from Rae1+/− x Bub3+/− intercrosses). Data shown are means and standard deviations derived from three independent MEF lines per genotype. (B) Chromosome analysis of MEFs (at passage 5) from 13.5-d-old embryos collected from Rae1+/− females that were fertilized by Bub3+/− males. Data shown are means and standard deviations derived from three independent MEF lines per genotype. We counted the chromosomes of 50 metaphase spreads per independent MEF line. 200 mitotic figures per MEF line were screened for premature sister chromatid separation (PMSCS). (C) Metaphase spreads showing sister chromatid cohesion in a wild-type MEF (left) and PMSCS in a double heterozygote MEF (right). (D) Comparison of chromosome number distributions between wild-type, single heterozygous, and double heterozygous MEF lines (combined values of each genotype). (E) Aneuploidy and PMSCS in primary splenocytes from 5-mo-old mice of various genotypes. Data shown are means and standard deviations derived from three mice per genotype. We counted the chromosomes of 50 metaphase spreads per mouse. 100 spreads per mouse were screened for PMSCS.
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fig8: Rae1 synergizes with Bub3 in the development of aneuploidy. (A) Growth curves of MEFs derived from wild-type, Rae1+/−, Bub3+/−, and Rae1+/−/Bub3+/− 13.5-d-old embryos (all harvested from Rae1+/− x Bub3+/− intercrosses). Data shown are means and standard deviations derived from three independent MEF lines per genotype. (B) Chromosome analysis of MEFs (at passage 5) from 13.5-d-old embryos collected from Rae1+/− females that were fertilized by Bub3+/− males. Data shown are means and standard deviations derived from three independent MEF lines per genotype. We counted the chromosomes of 50 metaphase spreads per independent MEF line. 200 mitotic figures per MEF line were screened for premature sister chromatid separation (PMSCS). (C) Metaphase spreads showing sister chromatid cohesion in a wild-type MEF (left) and PMSCS in a double heterozygote MEF (right). (D) Comparison of chromosome number distributions between wild-type, single heterozygous, and double heterozygous MEF lines (combined values of each genotype). (E) Aneuploidy and PMSCS in primary splenocytes from 5-mo-old mice of various genotypes. Data shown are means and standard deviations derived from three mice per genotype. We counted the chromosomes of 50 metaphase spreads per mouse. 100 spreads per mouse were screened for PMSCS.

Mentions: Next, we determined the effect of Rae1 haplo-insufficiency on chromosome number stability by performing chromosome counts on metaphase spreads from Rae1+/+ and Rae1+/− MEFs at passage 5. The average percentage of aneuploid metaphases was significantly higher in Rae1+/− MEFs than in Rae1+/+ MEFs (20 ± 2% vs. 9 ± 1%; Fig. 4 A; see also Fig. 8, B and D). In addition, Rae1+/− spreads showed a broader spectrum of abnormal chromosome numbers than Rae1+/+ spreads (Fig. 4 B; see also Fig. 8, B and D). Together, these findings show that loss of a single Rae1 allele leads to a significantly increased rate of chromosome missegregation.


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 synergizes with Bub3 in the development of aneuploidy. (A) Growth curves of MEFs derived from wild-type, Rae1+/−, Bub3+/−, and Rae1+/−/Bub3+/− 13.5-d-old embryos (all harvested from Rae1+/− x Bub3+/− intercrosses). Data shown are means and standard deviations derived from three independent MEF lines per genotype. (B) Chromosome analysis of MEFs (at passage 5) from 13.5-d-old embryos collected from Rae1+/− females that were fertilized by Bub3+/− males. Data shown are means and standard deviations derived from three independent MEF lines per genotype. We counted the chromosomes of 50 metaphase spreads per independent MEF line. 200 mitotic figures per MEF line were screened for premature sister chromatid separation (PMSCS). (C) Metaphase spreads showing sister chromatid cohesion in a wild-type MEF (left) and PMSCS in a double heterozygote MEF (right). (D) Comparison of chromosome number distributions between wild-type, single heterozygous, and double heterozygous MEF lines (combined values of each genotype). (E) Aneuploidy and PMSCS in primary splenocytes from 5-mo-old mice of various genotypes. Data shown are means and standard deviations derived from three mice per genotype. We counted the chromosomes of 50 metaphase spreads per mouse. 100 spreads per mouse were screened for PMSCS.
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Related In: Results  -  Collection

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fig8: Rae1 synergizes with Bub3 in the development of aneuploidy. (A) Growth curves of MEFs derived from wild-type, Rae1+/−, Bub3+/−, and Rae1+/−/Bub3+/− 13.5-d-old embryos (all harvested from Rae1+/− x Bub3+/− intercrosses). Data shown are means and standard deviations derived from three independent MEF lines per genotype. (B) Chromosome analysis of MEFs (at passage 5) from 13.5-d-old embryos collected from Rae1+/− females that were fertilized by Bub3+/− males. Data shown are means and standard deviations derived from three independent MEF lines per genotype. We counted the chromosomes of 50 metaphase spreads per independent MEF line. 200 mitotic figures per MEF line were screened for premature sister chromatid separation (PMSCS). (C) Metaphase spreads showing sister chromatid cohesion in a wild-type MEF (left) and PMSCS in a double heterozygote MEF (right). (D) Comparison of chromosome number distributions between wild-type, single heterozygous, and double heterozygous MEF lines (combined values of each genotype). (E) Aneuploidy and PMSCS in primary splenocytes from 5-mo-old mice of various genotypes. Data shown are means and standard deviations derived from three mice per genotype. We counted the chromosomes of 50 metaphase spreads per mouse. 100 spreads per mouse were screened for PMSCS.
Mentions: Next, we determined the effect of Rae1 haplo-insufficiency on chromosome number stability by performing chromosome counts on metaphase spreads from Rae1+/+ and Rae1+/− MEFs at passage 5. The average percentage of aneuploid metaphases was significantly higher in Rae1+/− MEFs than in Rae1+/+ MEFs (20 ± 2% vs. 9 ± 1%; Fig. 4 A; see also Fig. 8, B and D). In addition, Rae1+/− spreads showed a broader spectrum of abnormal chromosome numbers than Rae1+/+ spreads (Fig. 4 B; see also Fig. 8, B and D). Together, these findings show that loss of a single Rae1 allele leads to a significantly increased rate of chromosome missegregation.

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