Abstract
Nucleostemin (NS) is a nucleolar protein involved in the regulation of cell proliferation. Both overexpression and knockdown of NS increase the activity of the tumor suppressor protein p53, resulting in cell cycle arrest. In addition, NS regulates processing of pre-rRNA and consequently the level of total protein synthesis. Here, we describe a previously uncharacterized function of NS in the maintenance of the tripartite nucleolar structure as well as the integrity of small nucleolar ribonucleoproteins (snoRNPs). NS is also necessary to maintain the telomerase complex which shares common protein subunits with the H/ACA box snoRNPs. First, immunofluorescence microscopy and electron microscopy demonstrated that knockdown of NS disorganized the nucleolar architecture, in particular, the dense fibrillar component where snoRNPs are localized. Second, gel filtration chromatography and immunoprecipitation indicated that NS depletion leads to dissociation of the components of snoRNPs and the telomerase complex. Third, NS depletion reduced both telomerase activity and the cellular level of pseudouridine, an H/ACA snoRNP-mediated modification of rRNA and other RNAs that are important for their folding and stability. These morphological, biochemical and functional studies demonstrate that NS plays an important role to maintain nucleolar structure and function on a more fundamental level than previously thought.
Highlights
SEPTEMBER 25, 2009 VOLUME 284 NUMBER 39 upstream of the p53-mediated signaling pathway (1, 6 – 8)
In the current study we focused on an additional novel role of NS in the maintenance of the integrity of nucleolar structure and nucleolar RNA-protein complexes, such as small nucleolar ribonucleoproteins (snoRNPs) and the telomerase complex. snoRNPs can be divided into two major groups, C/D snoRNPs and H/ACA snoRNPs, based on the conserved motifs of the RNA component in each group (10 –12)
Nucleostemin and Integrity of snoRNPs and Telomerase catalytic domain of dyskerin, NHP2 directly interacts with snoRNAs [10]. snoRNPs are assembled as inactive presnoRNPs on nascent snoRNAs in the nucleoplasm and transported to Cajal bodies in the nucleus for maturation to functional complexes. snoRNPs are eventually transported to the nucleolus and function within this structure [10]
Summary
Cell Culture and NS Knockdown—HeLa cells were cultured in Minimum Essential Medium with 10% fetal bovine serum (FBS) supplemented with non-essential amino acids and sodium pyruvate. The cells were incubated with primary and secondary antibodies diluted in Washing Solution for 1 h each and counterstained with Topro 3 (Invitrogen) for 15 min. Cells were washed in PBS and fixed in 1% glutaraldehyde and 3% formaldehyde in 0.1 M phosphate buffer, pH 7.4. 100-l cytoplasmic buffer (10 mM HEPES pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.03% Nonidet P-40, 0.2 mM phenylmethylsulfonyl fluoride, 2 mM leupeptin, and 1.5 mM pepstatin A) was added to 1 ϫ 107 cells, and the cells were incubated on ice for 20 min. The pellet was resuspended in 50-l nuclear buffer (10 mM HEPES pH 7.4, 420 mM NaCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.2 mM EDTA, 25% glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 2 mM leupeptin, and 1.5 mM pepstatin A) and incubated on ice for 20 min. Primer sequences used for qRT-PCR combined and resolved with a Superdex 200 gel filtration col-
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