Nucleolar protein Nop25 is involved in nucleolar architecture

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Abstract

Nop25 is a putative RNA binding nucleolar protein associated with rRNA transcription. The present study was undertaken to determine the crucial function of Nop25 in the nucleolus. When N-terminal amino acids of Nop25 were overexpressed as a dominant negative effector in cells, the nucleolus was fragmented into small components. Knockdown of Nop25 by RNA interference also induced drastic nucleolar fragmentation. These results suggest that Nop25 is involved in nucleolar architecture and maintenance. Although nucleolar fragmentation induced the dispersion of nucleolar proteins from the nucleolus, it did not lead to cell cycle arrest or apoptosis, suggesting no effect of nucleolar fragmentation on the transcription and processing of rRNA molecules and subsequent ribosome biogenesis. Studies on Nop25 may provide new information on nucleolar organization related to nucleolar architecture and function.

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Materials and methods

Chemicals. A eukaryotic expression vector pEGFP-C1 was purchased from Clontech (Palo Alto, CA). Mouse monoclonal antibody against nucleophosmin and Cy3-conjugated anti-rabbit or mouse IgG were obtained from Lab Vision (Fermont, CA) and Chemicon (Temecula, CA), respectively. Rabbit polyclonal antibody against rat Nop25 was provided by Trans Genic Inc. (Kumamoto, Japan). Hoechst 33258 was purchased from Dojindo (Kumamoto, Japan).

Cell cultures. COS7 or HeLa cells were maintained in Dulbecco’s

Overexpression of N-terminal fragment of Nop25 induced nucleolus fragmentation

We mapped the nucleolar targeting sequence of Nop25. Deletion experiments have demonstrated that 70 amino acids at the N-terminus might play a role in targeting Nop25 into the nucleus [2]. The N-terminus contains many basic residues including lysine and arginine (Fig. 1A). When the 70 amino acids were fused with a GFP and expressed in cells (Fig. 1B, GFP-N70), the subcellular localization of GFP-N70 protein was different from that of GFP-Nop25 fusion protein (Fig. 1C, GFP-N70 vs. GFP-Nop25).

Discussion

Nucleolar disassembly and reassembly were observed by cytologists as early as the eighteenth century [15]. Although the molecular mechanisms regulating these phenomena remain largely unknown, our understanding of the mechanisms has increased tremendously [15]. The nucleolus is reversibly disassembled during mitosis, while it is irreversibly disassembled in apoptosis [14], [16]. During mitosis, nucleolar disassembly occurs following mitosis-specific phosphorylation of Pol I transcription factors

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