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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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HA–Rae1 expression restores mitotic checkpoint activation in both Rae1 and Bub3 haplo-insufficient cells. (A) Mitotic index of nocodazole-treated Rae1+/−/empty vector (n = 2) and Rae1+/−/HA–Rae1 (n = 2) cell lines. (B) Mitotic index of nocodazole-treated Bub3+/−/empty vector (n = 2) and Bub3+/−/HA–Rae1 (n = 2) cell lines. Note that HA–Rae1-expressing cultures accumulate mitotic cells over time, illustrating that their mitotic checkpoint activity is restored.
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fig7: HA–Rae1 expression restores mitotic checkpoint activation in both Rae1 and Bub3 haplo-insufficient cells. (A) Mitotic index of nocodazole-treated Rae1+/−/empty vector (n = 2) and Rae1+/−/HA–Rae1 (n = 2) cell lines. (B) Mitotic index of nocodazole-treated Bub3+/−/empty vector (n = 2) and Bub3+/−/HA–Rae1 (n = 2) cell lines. Note that HA–Rae1-expressing cultures accumulate mitotic cells over time, illustrating that their mitotic checkpoint activity is restored.

Mentions: Next, we cloned HA epitope–tagged mouse Rae1 cDNA into the pMSCV-Puro retroviral vector and used it to test whether ectopic expression of Rae1 would be able to correct for (a) Rae1 haplo-insufficiency (which would be expected if the mitotic phenotype is a direct consequence of the Rae1 mutation) and (b) Bub3+/− haplo-insufficiency (which would indicate that Rae1 has functional overlap with Bub3). We introduced the HA–Rae1 expression vector into two Rae1+/− and two Bub3+/− MEF lines by retroviral gene transfer (Kasper et al., 1999), selected the cultures for puromycin-resistant cells, and tested these cells for their ability to activate the mitotic checkpoint in response to nocodazole-induced spindle damage. Consistent with a reestablishment of normal mitotic checkpoint activity, the mitotic index of HA–Rae1-expressing Rae1+/− cultures showed a similar increase as Rae1+/+ cultures (Fig. 7 A). As expected, Rae1+/− cells transduced with empty pMSCV-Puro vector remained unable to sustain their mitotic checkpoint activity. Strikingly, HA–Rae1 was also capable of restoring the mitotic checkpoint defect caused by reduced Bub3 activity (Fig. 7 B). Thus, Rae1 can compensate for Bub3-mediated mitotic checkpoint activity that is affected by limited availability of Bub3 molecules.


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)

HA–Rae1 expression restores mitotic checkpoint activation in both Rae1 and Bub3 haplo-insufficient cells. (A) Mitotic index of nocodazole-treated Rae1+/−/empty vector (n = 2) and Rae1+/−/HA–Rae1 (n = 2) cell lines. (B) Mitotic index of nocodazole-treated Bub3+/−/empty vector (n = 2) and Bub3+/−/HA–Rae1 (n = 2) cell lines. Note that HA–Rae1-expressing cultures accumulate mitotic cells over time, illustrating that their mitotic checkpoint activity is restored.
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Related In: Results  -  Collection

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fig7: HA–Rae1 expression restores mitotic checkpoint activation in both Rae1 and Bub3 haplo-insufficient cells. (A) Mitotic index of nocodazole-treated Rae1+/−/empty vector (n = 2) and Rae1+/−/HA–Rae1 (n = 2) cell lines. (B) Mitotic index of nocodazole-treated Bub3+/−/empty vector (n = 2) and Bub3+/−/HA–Rae1 (n = 2) cell lines. Note that HA–Rae1-expressing cultures accumulate mitotic cells over time, illustrating that their mitotic checkpoint activity is restored.
Mentions: Next, we cloned HA epitope–tagged mouse Rae1 cDNA into the pMSCV-Puro retroviral vector and used it to test whether ectopic expression of Rae1 would be able to correct for (a) Rae1 haplo-insufficiency (which would be expected if the mitotic phenotype is a direct consequence of the Rae1 mutation) and (b) Bub3+/− haplo-insufficiency (which would indicate that Rae1 has functional overlap with Bub3). We introduced the HA–Rae1 expression vector into two Rae1+/− and two Bub3+/− MEF lines by retroviral gene transfer (Kasper et al., 1999), selected the cultures for puromycin-resistant cells, and tested these cells for their ability to activate the mitotic checkpoint in response to nocodazole-induced spindle damage. Consistent with a reestablishment of normal mitotic checkpoint activity, the mitotic index of HA–Rae1-expressing Rae1+/− cultures showed a similar increase as Rae1+/+ cultures (Fig. 7 A). As expected, Rae1+/− cells transduced with empty pMSCV-Puro vector remained unable to sustain their mitotic checkpoint activity. Strikingly, HA–Rae1 was also capable of restoring the mitotic checkpoint defect caused by reduced Bub3 activity (Fig. 7 B). Thus, Rae1 can compensate for Bub3-mediated mitotic checkpoint activity that is affected by limited availability of Bub3 molecules.

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