Human centromere chromatin protein hMis12, essential for equal segregation, is independent of CENP-A loading pathway.
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.
Goshima
Gohta
G
COE Research Project, Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
Kiyomitsu
Tomomi
T
Yoda
Kinya
K
Yanagida
Mitsuhiro
M
D000595:Amino Acid Sequence; D001324:Autoantigens; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D002503:Centromere / Q000378:metabolism; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D020090:Chromosome Segregation; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D018386:Kinetochores / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008677:Metaphase; D008938:Mitosis; D008954:Models, Biological; D008969:Molecular Sequence Data; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection
D001324:Autoantigens; C000619700:CENPA protein, human; D018797:Cell Cycle Proteins; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D008164:Luminescent Proteins; C120362:MTW1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C060905:centromere protein C; D049452:Green Fluorescent Proteins