Abstract
CENP-W was originally identified as a putative oncogene, cancer-upregulated gene 2 (CUG2) that was commonly up-regulated in many cancer tissues. Recently, CENP-W has also been identified as a new centromeric component that interacts with CENP-T. As a complex with CENP-T, CENP-W plays crucial roles in assembly of the functional kinetochore complex. In this study, the subnuclear localization of CENP-W was extensively analyzed using various approaches. We found that ectopically expressed CENP-W primarily accumulated in the nucleolus and remained substantially associated with the nucleolus in stable cells. The following fractionation study also showed that CENP-W is associated with RNA as well as DNA. Moreover, a considerable amount of CENP-W was found in the nuclear mesh-like structure, nuclear matrix, possibly indicating that CENP-W participates in diverse subnuclear activities. Finally, biochemical affinity binding analysis revealed that CENP-W specifically interacts with the nucleolar phosphoprotein, nucleophosmin (B23). Depletion of cellular B23 by siRNA treatment induced a dramatic decrease of CENP-W stability and severe mislocalization during prophase. Our data proposed that B23 may function in the assembly of the kinetochore complex by interacting with CENP-W during interphase.
Highlights
The nucleolus is one of the most observed amorphous subnuclear structures in mammalian cells during interphase [4]
CENP-W is initially identified as cancer-upregulated gene 2 (CUG2),3 which is commonly up-regulated in various types of human cancers and highly oncogenic when expressed in mouse fibroblast cell line [19]
CENP-W was originally identified as a putative oncogene that is up-regulated in many human cancer tissues [19]
Summary
The nucleolus is one of the most observed amorphous subnuclear structures in mammalian cells during interphase [4]. Because the subnuclear distribution of CENP-W in stable cells differed from that of the transiently expressed protein, we traced the localization of FLAG-CENP-W over time after transfection in HeLa cells using immunofluorescence microscopy.
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