Novel Role of Nucleostemin in the Maintenance of Nucleolar Architecture and Integrity of Small Nucleolar Ribonucleoproteins and the Telomerase Complex

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.

Distinct genome protective vs. ribosome synthetic functions of the paralogous nucleolar proteins nucleostemin and GNL3L

“…However, cells in which nucleostemin had been knocked down for 6 days displayed a substantial reduction (75%) in their nucleoplasmic 5-EU differential signal compared with siScr-treated cells. Finally, in contrast to a previous report (Romanova et al, 2009b) showing that nucleostemin depletion causes a dispersion of the dense fibrillar component (DFC) of nucleolar structure, which is defined by the protein fibrillarin, we found no discernible changes in the fibrillarin-labeled DFC structure in cells in which nucleostemin was knocked down for 2 days (Fig. 3C1), but a disorganized DFC structure was apparent in some cells after a 6-day knockdown (Fig.…”

“…In this respect, it is noteworthy that effects on ribosome synthesis have been observed following two rounds of RNAi-mediated nucleostemin knockdown over a period of 5 days (Romanova et al, 2009a;Romanova et al, 2009b). Motivated by the recent discovery of a role for nucleostemin in protecting telomeres and the genome integrity of stem and progenitor cells (Hsu et al, 2012;Lin et al, 2013;Meng et al, 2013;Yamashita et al, 2013), we began to consider whether the accumulation of DNA damage, rather than impaired ribosome production, is actually the initial event following nucleostemin depletion.…”

Dynamic and unique nucleolar microenvironment revealed by fluorescence correlation spectroscopy

“…Previous studies based on fluorescence observations have demonstrated that various nucleolar proteins, such as fibrillarin and nucleophosmin (B23), tagged to fluorescent proteins are functional and can be used as markers for observing changes in morphology of the nucleolus in live cells. In addition to fibrillarin and B23, NS, which is a key nucleolar protein in eukaryotes, has multiple functions and also contributes to the nucleolus architecture (12,13,36). It has been demonstrated that the different mobility of nucleolar proteins and the exchange rate between the nucleoplasm and the nucleolus under normal and ActDtreated conditions are related to their functional roles in ribosome biogenesis (13).…”

The dynamics of the alternatively spliced NOL7 gene products and role in nucleolar architecture

“…siRNA to nucleophosmin caused major distortion in nuclear and nucleolar structure. 45,46 siRNA to Nop25 caused nucleolar fragmentation. 47 Nucleostemin knockdown caused cell cycle arrest 48,49 and modified the internal pattern of the DFC, causing the loosening of snoRNP clusters.…”

“…47 Nucleostemin knockdown caused cell cycle arrest 48,49 and modified the internal pattern of the DFC, causing the loosening of snoRNP clusters. 46 USP36 depletion GAA TTC TTG TAC TTT AAC TTT CTT GGA; 1-193 aa, 581 bp, anti-sense: ATA GAA TTC ATG AAG GCT TGT GCT GCT TGT;…”