Nucleostemin is a nucleolar protein widely expressed in proliferating cells. Nucleostemin is involved in the regulation of cell proliferation, and both depletion and overexpression of nucleostemin induce cell cycle arrest through the p53 signaling pathway. Although the presence of p53-independent functions of nucleostemin has been previously suggested, the identities of these additional functions remained to be investigated. Here, we show that nucleostemin has a novel role as an integrated component of ribosome biogenesis, particularly pre-rRNA processing. Nucleostemin forms a large protein complex (>700 kDa) that co-fractionates with the pre-60 S ribosomal subunit in a sucrose gradient. This complex contains proteins related to prerRNA processing, such as Pes1, DDX21, and EBP2, in addition to several ribosomal proteins. We show that the nucleolar retention of DDX21 and EBP2 is dependent on the presence of nucleostemin in the nucleolus. Furthermore, the knockdown of nucleostemin delays the processing of 32 S pre-rRNA into 28 S rRNA. This is accompanied by a substantial decrease of protein synthesis as well as the levels of rRNAs and some mRNAs. In addition, overexpressed nucleostemin significantly promotes the processing of 32 S pre-rRNA. Collectively, these biochemical and functional studies demonstrate a novel role of nucleostemin in ribosome biogenesis. This is a key aspect of the role of nucleostemin in regulating cell proliferation. Nucleostemin (NS)2 is a nucleolar protein preferentially expressed in actively proliferating cells. The structure of NS is characterized by two GTP-binding domains, which are involved in the regulation of its dynamic shuttling between the nucleolus and nucleoplasm (1). NS was originally identified as a nucleolar protein prominently expressed in rat neural stem cells and down-regulated during differentiation of these cells in vitro (2). The same authors also found that NS is widely expressed in neural precursor cells in early mouse embryos as well as in a variety of cancer cells and stem cells, including embryonic stem cells and a hematopoietic stem cell-enriched fraction. NS is generally down-regulated in the early stage of differentiation before exit from the cell cycle. In addition, knockdown of NS significantly inhibits proliferation of cortical stem cells and cancer cells. These initial observations led to suggestions that NS is involved in multipotency in stem cells as well as in the regulation of cancer and stem cell proliferation (2).Recent work, however, has demonstrated that NS is in fact widely expressed in many types of normal proliferating cells at levels similar to those in malignant cells. For instance, NS is expressed in normal kidney cells and renal carcinoma cells at comparable levels as detected in histological sections (3). The expression of NS is significantly up-regulated when normal T lymphocytes are activated by concanavalin A (3) and when bone marrow stem cells are stimulated by fibroblast growth factor 2 (4). Cells in NS-null mouse embryos fail to enter ...

Object The accurate placement of cerebral ventricular shunt catheters in hydrocephalus is an important clinical problem. Malfunction of shunts remains their most common complication and greatest liability, and the influence of catheter position on shunt function remains poorly defined. The objectives of this study were as follows: 1) determine the accuracy of intraventricular catheter placement with respect to a historically favored target, defined as a 1-cm radius sphere at the anterior lip of the ipsilateral foramen of Monro; 2) confirm that this target represents a satisfactory site for frontal and occipital catheter placement by examining whether inaccuracy is associated with more shunt failures; and 3) determine whether catheter trajectory, use of image confirmation, or other factors are associated with either the accuracy or the longevity of shunts. Methods A retrospective cohort analysis was conducted on 236 patients with 426 ventricular shunts placed or revised at the University of Minnesota over a 10-year period. Results Accuracy of shunt placement was optimal in 43.9% of patients and suboptimal or poor in 56.1% of patients. Time to failure was significantly affected by the accuracy of catheter placement with respect to the ipsilateral foramen of Monro, with a 57% higher risk of failure with suboptimal placement (hazard ratio [HR] 1.57, 95% CI 1.26–1.96; p < 0.001) and a 66% higher risk with poor placement (HR 1.66, 95% CI 1.45–1.89; p < 0.001) relative to optimal placement. The odds of highly suboptimal or unacceptable placement were significantly increased by lack of any intraoperative imaging (OR 5.89, 95% CI 2.36–14.65; p < 0.001). Use of a nonfrontal posterior trajectory also showed a trend toward poor placement (OR 1.64, p = 0.138). Conclusions The historical target for catheter tip placement within 1 cm of the foramen of Monro in the ipsilateral lateral ventricle was associated with significantly longer revision-free survival compared with other locations. This effect remained significant after adjusting for age and whether there was a prior history of shunting. The accuracy of catheter placement in both pediatric and adult patients was strongly associated with use of intraoperative fluoroscopic confirmation. In analyses comparing intraoperative fluoroscopy and no imaging, there was a non–statistically significant difference in the 3-year time to failure, but the worst-case scenario of catastrophic short-term failure was almost completely avoided with fluoroscopy. The authors conclude that accuracy of placement is critical for shunt survival, and that use of intraoperative imaging confirmation may optimize outcomes by avoiding the majority of unacceptable placements.

The transcription factors Oct4 and Nanog are essential for the maintenance of an undifferentiated and pluripotent state in early embryonic cells, embryonic stem cells and embryonal carcinoma cells in humans and mice. These factors are co-localized to promoters of more than 300 genes, and synergistically regulate their activities. Currently, the molecular interaction between these two factors has not been well-characterized. During attempts to co-immunoprecipitate Oct4 and Nanog we found that cross-linking with dithiobis[succinimidylpropionate] was necessary to maintain their interaction. This result was supported by gel filtration analysis. Surprisingly, formaldehyde, a cross-linker commonly used during chromatin immunoprecipitation of Oct4 and Nanog, did not preserve the complex. Our findings demonstrate the effectiveness of using DSP to mitigate the instability of the interaction between these two particular proteins. Additionally, this solution may potentially allow us to identify novel members of the Oct4-Nanog complex, leading to better understanding of the regulatory mechanisms behind pluripotency.

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.

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