RNAi knockdown of human kinetochore protein CENP-H.
The inner kinetochore protein complex binds to centromeres during the whole cell cycle. It serves as the basis for the binding of further kinetochore proteins during mitosis. CENP-H is one of the inner kinetochore proteins which is conserved amongst many eukaryotes. By specific RNAi knockdown, we reduced the CENP-H protein level in human HEp-2 cells down to less than 5% of its normal value. In these CENP-H knocked-down cells, we observed severe mitotic phenotypes like misaligned chromosomes and multipolar spindles, however, no mitotic arrest. Strong reduction of CENP-H resulted in a slightly reduced CENP-C level at the kinetochores and normal localisation of hBubR1, indicating a functional mitotic checkpoint at the hBubR1 protein level. In CENP-H knocked-down human cells, the misaligned chromosomes contained only reduced levels of CENP-E. Our data clearly indicate that CENP-H has an important impact on the architecture and function of the human kinetochore complex.
Orthaus
Sandra
S
Department of Molecular Biology, FLI e.V., Beutenbergstrasse 11, D-07745 Jena, Germany.
Ohndorf
Sabine
S
Diekmann
Stephan
S
D002460:Cell Line; D002868:Chromosomal Proteins, Non-Histone / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018386:Kinetochores / Q000502:physiology*; D008938:Mitosis / Q000502:physiology*; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D034622:RNA Interference; D008941:Spindle Apparatus / Q000378:metabolism
C121933:CENPH protein, human; D002868:Chromosomal Proteins, Non-Histone; C068688:centromere protein E; D011494:Protein Kinases; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases