Biochemical and Biophysical Research Communications
Nucleolar protein Nop25 is involved in nucleolar architecture
Section snippets
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
References (19)
- et al.
Molecular cloning and characterization of Nop25, a novel nucleolar RNA binding protein highly, conserved in vertebrate species
Exp. Cell Res.
(2006) - et al.
Mapping a nucleolar targeting sequence of an RNA binding nucleolar protein, Nop25
Exp. Cell Res.
(2006) - et al.
New insights into nucleolar architecture and activity
Int. Rev. Cytol.
(2006) - et al.
Impaired ribosome biogenesis in Diamond-Blackfan anemia
Blood
(2007) - et al.
Requirement of the protein B23 for nucleolar disassembly induced by the FRGY2a family proteins
J. Biol. Chem.
(2006) - et al.
Functional expression of CCL6 by rat microglia: a possible role of CCL6 in cell–cell communication
J. Neuroimmunol.
(2005) - et al.
Genetic inactivation of the transcription factor TIF-IA leads to nucleolar disruption, cell cycle arrest, and p53-mediated apoptosis
Mol. Cell
(2005) Cell and molecular biology of nucleolar assembly and disassembly
Int. Rev. Cytol.
(2004)The plurifunctional nucleolus
Nucleic Acids Res.
(1998)