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Human centromere chromatin protein hMis12, essential for equal segregation, is independent of CENP-A loading pathway.

Goshima G, Kiyomitsu T, Yoda K, Yanagida M - J. Cell Biol. (2003)

Bottom Line: RNA interference (RNAi) analysis of HeLa cells shows that the reduced hMis12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei without mitotic delay, while CENP-A is located at kinetochores.RNAi for hMis6, like that of a kinetochore kinesin CENP-E, induces mitotic arrest.Kinetochore localization of hMis12 is unaffected by CENP-A RNAi, demonstrating an independent pathway of CENP-A in human kinetochores.

View Article: PubMed Central - PubMed

Affiliation: COE Research Project, Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.

ABSTRACT
Kinetochores are the chromosomal sites for spindle interaction and play a vital role for chromosome segregation. The composition of kinetochore proteins and their cellular roles are, however, poorly understood in higher eukaryotes. We identified a novel kinetochore protein family conserved from yeast to human that is essential for equal chromosome segregation. The human homologue hMis12 of yeast spMis12/scMtw1 retains conserved sequence features and locates at the kinetochore region indistinguishable from CENP-A, a centromeric histone variant. RNA interference (RNAi) analysis of HeLa cells shows that the reduced hMis12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei without mitotic delay, while CENP-A is located at kinetochores. Further, the metaphase spindle length is abnormally extended. Spindle checkpoint protein hMad2 temporally localizes at kinetochores at early mitotic stages after RNAi. The RNAi deficiency of CENP-A leads to a similar mitotic phenotype, but the kinetochore signals of other kinetochore proteins, hMis6 and CENP-C, are greatly diminished. RNAi for hMis6, like that of a kinetochore kinesin CENP-E, induces mitotic arrest. Kinetochore localization of hMis12 is unaffected by CENP-A RNAi, demonstrating an independent pathway of CENP-A in human kinetochores.

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Identification of hMis12. (A, top) Alignment of the NH2 terminus of Mis12-like proteins from S. pombe (Mis12), S. cerevisiae (Mtw1p), A. thaliana (atMis12; MOK9.12), M. musculus (mMis12; AK010995), and H. sapiens (hMis12; MGC2488). Identical amino acids are boxed and similar ones are hatched. The mutation site of S. pombe mis12-537 ts and S. cerevisiae mtw1-1 ts mutants is indicated (asterisk). (A, bottom) Schematic representation of the Mis12 family protein. The predicted coiled coil exists in the middle, while the two conserved regions (Block 1 and 2) are located in the NH2-terminal 100 aa. (B) Identification of hMis12 protein in HeLa cell extract by immunoblotting using affinity-purified anti-hMis12 antibody. (C) The COOH terminus of the hMis12 gene was tagged with V5His6, and plasmid carrying hMis12–V5His6 under the CMV promoter was transfected into HeLa cells (lanes 1 and 3). Vector plasmid pcDNA3.1-V5His6 was transfected as control (lanes 2 and 4). Extracts were prepared 24 h after transfection, and immunoblotting was performed using anti-hMis12 (lanes 1 and 2) or anti-V5 (lanes 3 and 4) antibodies. 28-kD bands were detected in lanes 1 and 3. (D) GFP was tagged to the NH2 terminus (GFP–hMis12) or COOH terminus (hMis12–GFP) of the hMis12 gene. The plasmid carrying GFP-tagged hMis12 under the CMV promoter was transfected into HeLa cells. Vector plasmid pEGFP-C1 was transfected as control. Extracts were prepared 12 and 24 h after transfection and immunoblotting was performed using anti-hMis12 antibody. 53-kD bands were additionally detected when GFP-tagged hMis12 was expressed. (E) No apparent change of hMis12 level in the cell cycle. HeLa cells were arrested at G1/S phase by double thymidine block (lane 3) or at M phase by nocodazole treatment (lane 2). Immunoblotting was performed using anti-hMis12, anti-cyclinB1, and anti-PSTAIR antibodies. CyclinB1 levels apparently increased in nocodazole-blocked M phase extracts. Extract from logarithmically growing cells was used as control (lane 1). (F) Kinetochore-like localization of hMis12. (F, top) Paraformaldehyde-fixed HeLa cells were stained by anti-hMis12 antibody (red) and anti-tubulin (green). hMis12 was localized on the M-phase condensed chromosomes showing many punctate signals. (F, bottom) No staining was observed on chromosomes when preimmune serum was used. DNA was stained by Hoechst 33342 (blue). Bar, 10 μm.
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fig1: Identification of hMis12. (A, top) Alignment of the NH2 terminus of Mis12-like proteins from S. pombe (Mis12), S. cerevisiae (Mtw1p), A. thaliana (atMis12; MOK9.12), M. musculus (mMis12; AK010995), and H. sapiens (hMis12; MGC2488). Identical amino acids are boxed and similar ones are hatched. The mutation site of S. pombe mis12-537 ts and S. cerevisiae mtw1-1 ts mutants is indicated (asterisk). (A, bottom) Schematic representation of the Mis12 family protein. The predicted coiled coil exists in the middle, while the two conserved regions (Block 1 and 2) are located in the NH2-terminal 100 aa. (B) Identification of hMis12 protein in HeLa cell extract by immunoblotting using affinity-purified anti-hMis12 antibody. (C) The COOH terminus of the hMis12 gene was tagged with V5His6, and plasmid carrying hMis12–V5His6 under the CMV promoter was transfected into HeLa cells (lanes 1 and 3). Vector plasmid pcDNA3.1-V5His6 was transfected as control (lanes 2 and 4). Extracts were prepared 24 h after transfection, and immunoblotting was performed using anti-hMis12 (lanes 1 and 2) or anti-V5 (lanes 3 and 4) antibodies. 28-kD bands were detected in lanes 1 and 3. (D) GFP was tagged to the NH2 terminus (GFP–hMis12) or COOH terminus (hMis12–GFP) of the hMis12 gene. The plasmid carrying GFP-tagged hMis12 under the CMV promoter was transfected into HeLa cells. Vector plasmid pEGFP-C1 was transfected as control. Extracts were prepared 12 and 24 h after transfection and immunoblotting was performed using anti-hMis12 antibody. 53-kD bands were additionally detected when GFP-tagged hMis12 was expressed. (E) No apparent change of hMis12 level in the cell cycle. HeLa cells were arrested at G1/S phase by double thymidine block (lane 3) or at M phase by nocodazole treatment (lane 2). Immunoblotting was performed using anti-hMis12, anti-cyclinB1, and anti-PSTAIR antibodies. CyclinB1 levels apparently increased in nocodazole-blocked M phase extracts. Extract from logarithmically growing cells was used as control (lane 1). (F) Kinetochore-like localization of hMis12. (F, top) Paraformaldehyde-fixed HeLa cells were stained by anti-hMis12 antibody (red) and anti-tubulin (green). hMis12 was localized on the M-phase condensed chromosomes showing many punctate signals. (F, bottom) No staining was observed on chromosomes when preimmune serum was used. DNA was stained by Hoechst 33342 (blue). Bar, 10 μm.

Mentions: Fission yeast spMis12 and budding yeast Mtw1 contain the conserved 100-aa NH2 terminus, which consists of two highly conserved regions, block 1 and 2 (Fig. 1 A), immediately followed by the 50-aa presumed coiled coil. By BLAST search, however, homologous sequences with the meaningful E-value were not found in the genome of higher eukaryotes. We therefore employed the Block Maker (Henikoff et al., 1998; http://www.blocks.fhcrc.org/blocks/blockmkr/make_blocks.html) and MAST (Multiple Alignment and Search Tool; Bailey and Gribskov, 1998) for searching for similar genes under the conditions of two block sequences and one coiled-coil region. Three fungal homologues (Candida albicans, Aspergillus nidulans, and Magnaporthe grisea) and one Arabidopsis thaliana homologue (atMis12, MOK9.12; E-value = 0.058) have been obtained. Arabidopsis atMis12 also contained the 50-aa coiled coil after block 2. A similar gene was found in the bean Glycine max (sp43a06.y1). Using these five fungal and two plant Mis12 homologues for the Block Maker and MAST searching, we were able to obtain putative homologues of human (MGC2488; E-value = 1) and mouse (AK010995; E-value = 8.9), as shown in Fig. 1 A. Although the above E-values are much higher than those between Mis12 and Mtw1 (9e-08), all of them were in the range of 25–35 kD, containing two similar blocks and the 50-aa coiled coil. Databases of rat, bovine, and frog displayed proteins highly similar to hMis12. In fly, however, no putative homologue was found. Caenorhabditis elegans contained a potential homologue (Y47G6A.24; E-value = 8.1; RNAi of this gene leads to embryonic lethality; Maeda et al., 2001) but the similarity was low compared with other Mis12 homologues.


Human centromere chromatin protein hMis12, essential for equal segregation, is independent of CENP-A loading pathway.

Goshima G, Kiyomitsu T, Yoda K, Yanagida M - J. Cell Biol. (2003)

Identification of hMis12. (A, top) Alignment of the NH2 terminus of Mis12-like proteins from S. pombe (Mis12), S. cerevisiae (Mtw1p), A. thaliana (atMis12; MOK9.12), M. musculus (mMis12; AK010995), and H. sapiens (hMis12; MGC2488). Identical amino acids are boxed and similar ones are hatched. The mutation site of S. pombe mis12-537 ts and S. cerevisiae mtw1-1 ts mutants is indicated (asterisk). (A, bottom) Schematic representation of the Mis12 family protein. The predicted coiled coil exists in the middle, while the two conserved regions (Block 1 and 2) are located in the NH2-terminal 100 aa. (B) Identification of hMis12 protein in HeLa cell extract by immunoblotting using affinity-purified anti-hMis12 antibody. (C) The COOH terminus of the hMis12 gene was tagged with V5His6, and plasmid carrying hMis12–V5His6 under the CMV promoter was transfected into HeLa cells (lanes 1 and 3). Vector plasmid pcDNA3.1-V5His6 was transfected as control (lanes 2 and 4). Extracts were prepared 24 h after transfection, and immunoblotting was performed using anti-hMis12 (lanes 1 and 2) or anti-V5 (lanes 3 and 4) antibodies. 28-kD bands were detected in lanes 1 and 3. (D) GFP was tagged to the NH2 terminus (GFP–hMis12) or COOH terminus (hMis12–GFP) of the hMis12 gene. The plasmid carrying GFP-tagged hMis12 under the CMV promoter was transfected into HeLa cells. Vector plasmid pEGFP-C1 was transfected as control. Extracts were prepared 12 and 24 h after transfection and immunoblotting was performed using anti-hMis12 antibody. 53-kD bands were additionally detected when GFP-tagged hMis12 was expressed. (E) No apparent change of hMis12 level in the cell cycle. HeLa cells were arrested at G1/S phase by double thymidine block (lane 3) or at M phase by nocodazole treatment (lane 2). Immunoblotting was performed using anti-hMis12, anti-cyclinB1, and anti-PSTAIR antibodies. CyclinB1 levels apparently increased in nocodazole-blocked M phase extracts. Extract from logarithmically growing cells was used as control (lane 1). (F) Kinetochore-like localization of hMis12. (F, top) Paraformaldehyde-fixed HeLa cells were stained by anti-hMis12 antibody (red) and anti-tubulin (green). hMis12 was localized on the M-phase condensed chromosomes showing many punctate signals. (F, bottom) No staining was observed on chromosomes when preimmune serum was used. DNA was stained by Hoechst 33342 (blue). Bar, 10 μm.
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fig1: Identification of hMis12. (A, top) Alignment of the NH2 terminus of Mis12-like proteins from S. pombe (Mis12), S. cerevisiae (Mtw1p), A. thaliana (atMis12; MOK9.12), M. musculus (mMis12; AK010995), and H. sapiens (hMis12; MGC2488). Identical amino acids are boxed and similar ones are hatched. The mutation site of S. pombe mis12-537 ts and S. cerevisiae mtw1-1 ts mutants is indicated (asterisk). (A, bottom) Schematic representation of the Mis12 family protein. The predicted coiled coil exists in the middle, while the two conserved regions (Block 1 and 2) are located in the NH2-terminal 100 aa. (B) Identification of hMis12 protein in HeLa cell extract by immunoblotting using affinity-purified anti-hMis12 antibody. (C) The COOH terminus of the hMis12 gene was tagged with V5His6, and plasmid carrying hMis12–V5His6 under the CMV promoter was transfected into HeLa cells (lanes 1 and 3). Vector plasmid pcDNA3.1-V5His6 was transfected as control (lanes 2 and 4). Extracts were prepared 24 h after transfection, and immunoblotting was performed using anti-hMis12 (lanes 1 and 2) or anti-V5 (lanes 3 and 4) antibodies. 28-kD bands were detected in lanes 1 and 3. (D) GFP was tagged to the NH2 terminus (GFP–hMis12) or COOH terminus (hMis12–GFP) of the hMis12 gene. The plasmid carrying GFP-tagged hMis12 under the CMV promoter was transfected into HeLa cells. Vector plasmid pEGFP-C1 was transfected as control. Extracts were prepared 12 and 24 h after transfection and immunoblotting was performed using anti-hMis12 antibody. 53-kD bands were additionally detected when GFP-tagged hMis12 was expressed. (E) No apparent change of hMis12 level in the cell cycle. HeLa cells were arrested at G1/S phase by double thymidine block (lane 3) or at M phase by nocodazole treatment (lane 2). Immunoblotting was performed using anti-hMis12, anti-cyclinB1, and anti-PSTAIR antibodies. CyclinB1 levels apparently increased in nocodazole-blocked M phase extracts. Extract from logarithmically growing cells was used as control (lane 1). (F) Kinetochore-like localization of hMis12. (F, top) Paraformaldehyde-fixed HeLa cells were stained by anti-hMis12 antibody (red) and anti-tubulin (green). hMis12 was localized on the M-phase condensed chromosomes showing many punctate signals. (F, bottom) No staining was observed on chromosomes when preimmune serum was used. DNA was stained by Hoechst 33342 (blue). Bar, 10 μm.
Mentions: Fission yeast spMis12 and budding yeast Mtw1 contain the conserved 100-aa NH2 terminus, which consists of two highly conserved regions, block 1 and 2 (Fig. 1 A), immediately followed by the 50-aa presumed coiled coil. By BLAST search, however, homologous sequences with the meaningful E-value were not found in the genome of higher eukaryotes. We therefore employed the Block Maker (Henikoff et al., 1998; http://www.blocks.fhcrc.org/blocks/blockmkr/make_blocks.html) and MAST (Multiple Alignment and Search Tool; Bailey and Gribskov, 1998) for searching for similar genes under the conditions of two block sequences and one coiled-coil region. Three fungal homologues (Candida albicans, Aspergillus nidulans, and Magnaporthe grisea) and one Arabidopsis thaliana homologue (atMis12, MOK9.12; E-value = 0.058) have been obtained. Arabidopsis atMis12 also contained the 50-aa coiled coil after block 2. A similar gene was found in the bean Glycine max (sp43a06.y1). Using these five fungal and two plant Mis12 homologues for the Block Maker and MAST searching, we were able to obtain putative homologues of human (MGC2488; E-value = 1) and mouse (AK010995; E-value = 8.9), as shown in Fig. 1 A. Although the above E-values are much higher than those between Mis12 and Mtw1 (9e-08), all of them were in the range of 25–35 kD, containing two similar blocks and the 50-aa coiled coil. Databases of rat, bovine, and frog displayed proteins highly similar to hMis12. In fly, however, no putative homologue was found. Caenorhabditis elegans contained a potential homologue (Y47G6A.24; E-value = 8.1; RNAi of this gene leads to embryonic lethality; Maeda et al., 2001) but the similarity was low compared with other Mis12 homologues.

Bottom Line: RNA interference (RNAi) analysis of HeLa cells shows that the reduced hMis12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei without mitotic delay, while CENP-A is located at kinetochores.RNAi for hMis6, like that of a kinetochore kinesin CENP-E, induces mitotic arrest.Kinetochore localization of hMis12 is unaffected by CENP-A RNAi, demonstrating an independent pathway of CENP-A in human kinetochores.

View Article: PubMed Central - PubMed

Affiliation: COE Research Project, Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.

ABSTRACT
Kinetochores are the chromosomal sites for spindle interaction and play a vital role for chromosome segregation. The composition of kinetochore proteins and their cellular roles are, however, poorly understood in higher eukaryotes. We identified a novel kinetochore protein family conserved from yeast to human that is essential for equal chromosome segregation. The human homologue hMis12 of yeast spMis12/scMtw1 retains conserved sequence features and locates at the kinetochore region indistinguishable from CENP-A, a centromeric histone variant. RNA interference (RNAi) analysis of HeLa cells shows that the reduced hMis12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei without mitotic delay, while CENP-A is located at kinetochores. Further, the metaphase spindle length is abnormally extended. Spindle checkpoint protein hMad2 temporally localizes at kinetochores at early mitotic stages after RNAi. The RNAi deficiency of CENP-A leads to a similar mitotic phenotype, but the kinetochore signals of other kinetochore proteins, hMis6 and CENP-C, are greatly diminished. RNAi for hMis6, like that of a kinetochore kinesin CENP-E, induces mitotic arrest. Kinetochore localization of hMis12 is unaffected by CENP-A RNAi, demonstrating an independent pathway of CENP-A in human kinetochores.

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