Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere.
The centromere is a complex structure, the components and assembly pathway of which remain inadequately defined. Here, we demonstrate that centromeric alpha-satellite RNA and proteins CENPC1 and INCENP accumulate in the human interphase nucleolus in an RNA polymerase I-dependent manner. The nucleolar targeting of CENPC1 and INCENP requires alpha-satellite RNA, as evident from the delocalization of both proteins from the nucleolus in RNase-treated cells, and the nucleolar relocalization of these proteins following alpha-satellite RNA replenishment in these cells. Using protein truncation and in vitro mutagenesis, we have identified the nucleolar localization sequences on CENPC1 and INCENP. We present evidence that CENPC1 is an RNA-associating protein that binds alpha-satellite RNA by an in vitro binding assay. Using chromatin immunoprecipitation, RNase treatment, and "RNA replenishment" experiments, we show that alpha-satellite RNA is a key component in the assembly of CENPC1, INCENP, and survivin (an INCENP-interacting protein) at the metaphase centromere. Our data suggest that centromere satellite RNA directly facilitates the accumulation and assembly of centromere-specific nucleoprotein components at the nucleolus and mitotic centromere, and that the sequestration of these components in the interphase nucleolus provides a regulatory mechanism for their timely release into the nucleoplasm for kinetochore assembly at the onset of mitosis.
Wong
Lee H
LH
Chromosome and Chromatin Research Laboratory, Murdoch Children's Research Institute & Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria, Australia. lee.wong@mcri.edu.au
Brettingham-Moore
Kate H
KH
Chan
Lyn
L
Quach
Julie M
JM
Anderson
Melissa A
MA
Northrop
Emma L
EL
Hannan
Ross
R
Saffery
Richard
R
Shaw
Margaret L
ML
Williams
Evan
E
Choo
K H Andy
KH
D000595:Amino Acid Sequence; D001665:Binding Sites; D002466:Cell Nucleolus / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002503:Centromere / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006801:Humans; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism*; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D002868:Chromosomal Proteins, Non-Histone; C083767:INCENP protein, human; D011993:Recombinant Fusion Proteins; C060905:centromere protein C; D003609:Dactinomycin; D012313:RNA