Critical Role of Nucleostemin in Pre-rRNA Processing

Abstract

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 ...

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

“…Subsequent work on both nucleostemin and GNL3L (Lin et al, 2010;Ma and Pederson, 2007;Meng et al, 2011a;Meng et al, 2011b;Meng et al, 2008;Zhu et al, 2009;Zhu et al, 2006) drew attention to the involvement of both proteins in cell proliferation, although the direct roles of the proteins were not rigorously proven. At the same time, evidence appeared in Drosophila linking the invertebrate protein, GNL3, to ribosome biosynthesis (Rosby et al, 2009), and another report implicated mammalian nucleostemin in ribosome biosynthesis (Romanova et al, 2009a). It was against this background that we launched the present study.…”

“…Effects shown include a G1/S arrest (Dai et al, 2008;Ma and Pederson, 2007;Nikpour et al, 2009) 2009; Romanova et al, 2009a;Zhu et al, 2006). This discrepancy cannot be explained by the status of p53, Rb or p16 in the cell models used.…”

“…Most previous studies examined only the terminal results of nucleostemin gene knockout or knockdown, without resolving the temporal relationship of the events. This issue applies to both wholeorganism studies (Kudron and Reinke, 2008;Rosby et al, 2009) and to the nucleostemin-knockdown study of Romanova et al (Romanova et al, 2009a) in HeLa cells, in which cells were analyzed on the 5th day after two rounds of knockdown. Our timecourse analyses show that nucleostemin depletion triggers DNA damage and cell-cycle arrest shortly after the initiation of knockdown (,12 hours).…”

“…In C. elegans, loss of NST-1 decreases the levels of 18S and 26S rRNAs (Kudron and Reinke, 2008), and in Drosophila depletion of NS1 protein results in nucleolar accumulation of the large ribosomal subunit proteins L11 and L26 (Rosby et al, 2009). In mammalian cells, a potential role of nucleostemin in ribosomal synthesis was suggested by a study showing that prolonged knockdown of nucleostemin delayed the processing of 32S pre-rRNA to 28S ribosomal (r)RNA (Romanova et al, 2009a). Although these studies indicate that the loss of nucleostemin might eventually lead to the perturbation of ribosomes, they fail to establish a coherent mechanism or a direct target of nucleostemin action in the ribosomal-synthetic pathway.…”

“…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.…”