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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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Localization dependencies of kinetochore proteins hMis12, CENP-A, hMis6, CENP-C, and CENP-H in HeLa cells revealed by the RNAi method. (A) Results of intracellular localization of five kinetochore proteins in three different RNAi knockdowns are summarized. The localization of hMis12 and CENP-A is independent. (B) Localization dependency of kinetochore proteins in human and S. pombe is illustrated. The arrows indicate the requirement of functional kinetochore protein for proper localization of the downward proteins. In S. pombe, the localization of spMis12 and spCENP-A is independent, but the directionality for the requirements of Mis6 and CENP-A is different (Takahashi et al., 2000). The reason for this difference is unclear.
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fig8: Localization dependencies of kinetochore proteins hMis12, CENP-A, hMis6, CENP-C, and CENP-H in HeLa cells revealed by the RNAi method. (A) Results of intracellular localization of five kinetochore proteins in three different RNAi knockdowns are summarized. The localization of hMis12 and CENP-A is independent. (B) Localization dependency of kinetochore proteins in human and S. pombe is illustrated. The arrows indicate the requirement of functional kinetochore protein for proper localization of the downward proteins. In S. pombe, the localization of spMis12 and spCENP-A is independent, but the directionality for the requirements of Mis6 and CENP-A is different (Takahashi et al., 2000). The reason for this difference is unclear.

Mentions: A principal conclusion in the present study is that kinetochore localization dependency does not appear to exist between CENP-A and hMis12 in HeLa cells. The same independent behavior was found in fission yeast (Takahashi et al., 2000). Fission yeast and human Mis12 are localized in the kinetochores in CENP-A–knockdown cells. Fig. 8 summarizes the localization dependency of kinetochore proteins in HeLa cells revealed by this study. CENP-A localization does not require hMis6. Instead, the localization of hMis6 at the centromere needs both hMis12 and CENP-A. This is very different from the case of fission yeast in which spMis6 is essential for spCENP-A loading, and neither spCENP-A nor spMis12 is required for kinetochore localization of Mis6 (Goshima et al., 1999; Takahashi et al., 2000). The reason for this striking difference is unclear. In regard to CENP-C, its kinetochore localization depends on CENP-A, but not on hMis6, differing from the properties reported for chicken (Nishihashi et al., 2002). Dependency of localization for the case of CENP-C is not conserved, even among vertebrates. hMis12 is thus the first example that shows identical localization to CENP-A, but independently of the presence of functional CENP-A in multicellular organisms (Howman et al., 2000; Blower and Karpen, 2001; Oegema et al., 2001). This may suggest that CENP-A is not the sole inner chromatin core and that CENP-A RNAi does not necessarily result in the “kinetochore-” phenotype in humans, as hMis12 is still capable of being recruited to the kinetochore. Mis12 seems to form a loading pathway distinct from that of CENP-A. One may argue that a residual amount of CENP-A that could not be detected by immunofluorescence might be sufficient to allow localization of hMis12. This is always the potential problem in any kind of functional analyses of essential genes, even in the yeast gene disruption experiment, because 100% depletion of an essential protein from a cell is impossible (the yeast gene-disrupted spores often contain protein derived from zygotes). It is hence not completely ruled out that the residual CENP-A (10% or less) not detected by immunostaining may be sufficient to allow nearly full localization of hMis12. In the present study, the clear disappearance of CENP-C, CENP-H, and hMis6 from kinetochores after CENP-A RNAi strongly supports the model that hMis12 is classified into a functionally different group from the CENP-A pathway in the kinetochore assembly process.


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)

Localization dependencies of kinetochore proteins hMis12, CENP-A, hMis6, CENP-C, and CENP-H in HeLa cells revealed by the RNAi method. (A) Results of intracellular localization of five kinetochore proteins in three different RNAi knockdowns are summarized. The localization of hMis12 and CENP-A is independent. (B) Localization dependency of kinetochore proteins in human and S. pombe is illustrated. The arrows indicate the requirement of functional kinetochore protein for proper localization of the downward proteins. In S. pombe, the localization of spMis12 and spCENP-A is independent, but the directionality for the requirements of Mis6 and CENP-A is different (Takahashi et al., 2000). The reason for this difference is unclear.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172742&req=5

fig8: Localization dependencies of kinetochore proteins hMis12, CENP-A, hMis6, CENP-C, and CENP-H in HeLa cells revealed by the RNAi method. (A) Results of intracellular localization of five kinetochore proteins in three different RNAi knockdowns are summarized. The localization of hMis12 and CENP-A is independent. (B) Localization dependency of kinetochore proteins in human and S. pombe is illustrated. The arrows indicate the requirement of functional kinetochore protein for proper localization of the downward proteins. In S. pombe, the localization of spMis12 and spCENP-A is independent, but the directionality for the requirements of Mis6 and CENP-A is different (Takahashi et al., 2000). The reason for this difference is unclear.
Mentions: A principal conclusion in the present study is that kinetochore localization dependency does not appear to exist between CENP-A and hMis12 in HeLa cells. The same independent behavior was found in fission yeast (Takahashi et al., 2000). Fission yeast and human Mis12 are localized in the kinetochores in CENP-A–knockdown cells. Fig. 8 summarizes the localization dependency of kinetochore proteins in HeLa cells revealed by this study. CENP-A localization does not require hMis6. Instead, the localization of hMis6 at the centromere needs both hMis12 and CENP-A. This is very different from the case of fission yeast in which spMis6 is essential for spCENP-A loading, and neither spCENP-A nor spMis12 is required for kinetochore localization of Mis6 (Goshima et al., 1999; Takahashi et al., 2000). The reason for this striking difference is unclear. In regard to CENP-C, its kinetochore localization depends on CENP-A, but not on hMis6, differing from the properties reported for chicken (Nishihashi et al., 2002). Dependency of localization for the case of CENP-C is not conserved, even among vertebrates. hMis12 is thus the first example that shows identical localization to CENP-A, but independently of the presence of functional CENP-A in multicellular organisms (Howman et al., 2000; Blower and Karpen, 2001; Oegema et al., 2001). This may suggest that CENP-A is not the sole inner chromatin core and that CENP-A RNAi does not necessarily result in the “kinetochore-” phenotype in humans, as hMis12 is still capable of being recruited to the kinetochore. Mis12 seems to form a loading pathway distinct from that of CENP-A. One may argue that a residual amount of CENP-A that could not be detected by immunofluorescence might be sufficient to allow localization of hMis12. This is always the potential problem in any kind of functional analyses of essential genes, even in the yeast gene disruption experiment, because 100% depletion of an essential protein from a cell is impossible (the yeast gene-disrupted spores often contain protein derived from zygotes). It is hence not completely ruled out that the residual CENP-A (10% or less) not detected by immunostaining may be sufficient to allow nearly full localization of hMis12. In the present study, the clear disappearance of CENP-C, CENP-H, and hMis6 from kinetochores after CENP-A RNAi strongly supports the model that hMis12 is classified into a functionally different group from the CENP-A pathway in the kinetochore assembly process.

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.

Show MeSH