Oncogene (2010) 29, 4253–4260 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc REVIEW Ribosome biogenesis surveillance: probing the ribosomal protein-Mdm2-p53 pathway C Deisenroth1,2,4 and Y Zhang1,2,3,4 1Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 2Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 3Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA and 4Department of Radiation Oncology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA The dynamic processes of cell growth and cell division The Mdm2-p53 stress response pathway is an remain under constant surveillance. As one of the primary important regulator of cellular homeostasis. A variety ‘gatekeepers’ of the cell, p53 has a major role in sensing a of mitogenic and genotoxic stressors converge on this variety of stressors to maintain cellular homeostasis. pathway to elicit a protective p53-dependent stress Growth is driven by new protein synthesis, a process that response resulting in cell cycle arrest, apoptosis, DNA requires robust manufacture of ribosomes in the nucleolus. repair or replicative senescence (Levine et al., 2006). Ribosome biogenesis is a complex process comprising As the cellular ‘gatekeeper’, p53 remains at the pinnacle transcription, modification, and processing of ribosomal of cellular surveillance to regulate cell growth and RNA, production of ribosomal proteins (RPs) and proliferative homeostasis (Vogelstein et al., 2000). auxiliary factors, and coordinated assembly of ribo- Mdm2 is an E3 ubiquitin ligase that binds to p53 to nucleoprotein particles to produce mature ribosomes. As promote ubiquitination and degradation of the protein. the major function of the nucleolus, ribosome biogenesis As p53 is largely regulated at a posttranslational level, demands a considerable amount of resources and must be the primary mechanism for induced stability is mod- maintained in a coordinated manner to ensure fidelity of ifications to Mdm2–p53 interactions. There is a large the process. Perturbations to many aspects of ribosome array of regulatory factors that act on Mdm2 to regulate biogenesis are thought to contribute to ‘nucleolar stress’ p53 stability and function. One example is the INK4a/ and trigger a RP-Mdm2-p53 stress response pathway. In ARF locus, which encodes the alternative reading frame this review, we will clarify how disruption to three major (ARF) tumor suppressor; a small nucleolar protein. components of ribosome biogenesis can trigger nucleolar In response to oncogenic stress such as RAS or c-myc stress and activate p53, thereby lending support to a overexpression, ARF binds to and inhibits Mdm2- RP-Mdm2-p53 ribosome biogenesis surveillance pathway. mediated p53 ubiquitination and degradation (Sharp- Oncogene (2010) 29, 4253–4260; doi:10.1038/onc.2010.189; less, 2005). Mdm2 is also reported to be extensively published online 24 May 2010 modified through a number of posttranslational modifications including ubiquitination, sumoylation Keywords: ribosome biogenesis; ribosomal protein; and phosphorylation (Meek and Knippschild, 2003). Mdm2; p53; nucleolar stress Although DNA damage is known to activate ATM kinase-induced phosphorylation of p53, it also targets Mdm2 at ser395 for phosphorylation in vitro (Maya Introduction et al., 2001). Furthermore, two multisite phosphoryla- tion clusters were identified in vivo at the N-terminus of Cell growth and division have long been studied as two Mdm2, the region comprising the p53-binding domain inherently separate processes to understand the funda- and nuclear localization signal (Hay and Meek, 2000). mental mechanisms that drive these two systems. Hypophosphorylation of putative sites within the acidic However, consistent evidence has pointed toward domain of Mdm2 reduced or ablated the ability of the coupling of growth and division, suggesting the Mdm2 to degrade p53 (Blattner et al., 2002). Together, existence of molecular ties that bind these two processes. the evidence suggests that, like phosphorylation of p53, The signaling pathways that converge to connect protein Mdm2 phosphorylation inhibits Mdm2-directed turn- synthesis to regulation of the cell cycle have thus far over of p53. remained largely elusive. The latest players in Mdm2 regulation are a subset of ribosomal proteins (RPs) with well-established extra- Correspondence: Dr Y Zhang, Lineberger Comprehensive Cancer ribosomal function (Warner and McIntosh, 2009). Center and Department of Radiation Oncology-Box 7512, University The earliest evidence of RP interactions with Mdm2 of North Carolina at Chapel Hill, 101 Manning Drive, Chapel Hill, occurred with the report of RPL5 binding to Mdm2 in a NC 27514, USA. E-mail: [email protected] 5S ribosomal RNA (rRNA)-RPL5-Mdm2-p53 ribonu- Received 1 February 2010; revised 20 April 2010; accepted 22 April 2010; cleoprotein complex, but at the time, the meaning of published online 24 May 2010 such an interaction was unclear (Marechal et al., 1994). Ribosome biogenesis surveillance C Deisenroth and Y Zhang 4254 Nearly a decade later, in screens seeking out novel 1999), it stands to reason that the coordinated activities Mdm2 modulating proteins, the large subunit RPs of ribosome synthesis would be subject to extensive RPL5, RPL11 and RPL23 were all reported to bind to quality control surveillance. Mdm2, block the E3 ubiquitin ligase function of Mdm2, The site for ribosome synthesis occurs in the non- and promote p53 accumulation (Lohrum et al., 2003; membrane bound structure of the nucleolus. Nucleolar Zhang et al., 2003; Bhat et al., 2004; Dai and Lu, 2004; organization regions form around rDNA gene clusters Dai et al., 2004; Jin et al., 2004). Following these initial and are composed of a fibrillar center in which rDNA is reports, additional evidence subsequently was produced transcribed, a dense fibrillar component in which rRNA to support the roles of RPS7 (Chen et al., 2007; Zhu undergoes extensive modification and cleavage proces- et al., 2009), RPL26 (Ofir-Rosenfeld et al., 2008) and sing, and a granular component in which the bulk of the RPS3 (Yadavilli et al., 2009) as Mdm2-binding partners. RPs reside and assist in ribosomal subunit assembly. These studies served as the initial framework for Approximately 30% of the nucleolar proteome consists establishing an RP-Mdm2-p53 stress response pathway. of proteins involved in ribosome biogenesis, marking the In general, the current paradigm for ribosome nucleolus as the central location for ribosome assembly biogenesis, largely derived from studies in bacteria and (Boisvert et al., 2007). yeast systems, is the coordinated assembly of equimolar Many of the reports analyzing RP-Mdm2 binding concentrations of RPs and rRNA to generate mature have alluded to ‘nucleolar stress’ as the event responsible 80S polysomes that ensure adequate protein synthesis for inducing the RP-Mdm2-p53 stress response. In this and maintain cellular homeostasis. In humans, this context, ‘nucleolar stress’ specifically refers to perturba- process requires the activity of RNA polymerase I (PolI) tions of ribosome biogenesis and the subsequent break- to generate the 47S precursor rRNA from clusters of down of nucleolar structure, resulting in activation of ribosomal DNA (rDNA) tandem repeat genes. The p53. In part, these observations have led to the precursor is further processed to 18S, 5.8S and 28S hypothesis of the nucleolus as a central stress response rRNAs. The fourth rRNA, 5S, is transcribed separately regulator for p53 activation (Rubbi and Milner, 2003). by RNA polymerase III (PolIII) in the nucleus, exported One technique that has been used to induce nucleolar to the cytoplasm and finally imported to the nucleolus stress is inhibition of precursor rRNA synthesis using for incorporation into the large subunit (Szymanski low doses of actinomycin D (Bhat et al., 2004; Jin et al., et al., 2003). The pool of 79 RPs are actively transcribed 2004), 5-flourouracil (Sun et al., 2007) and myco- by RNA polymerase II, exported to the cytosol for phenolic acid (Sun et al., 2008). However, with regards to an translation and imported to the nucleolus for assembly RP-Mdm2-p53 pathway, it is imperative to identify the (Lempiainen and Shore, 2009). In addition, there are a natural biological mechanisms of nucleolar stress, suite of auxiliary factors that assist in the processing of particularly as they pertain to ribosome biogenesis. rRNA, assembly of the small and large subunits and In turn, identifying specific stressors of ribosome finally export and maturation of the functional ribo- assembly that induce the RP-Mdm2-p53 pathway some (Kressler et al., 1999; Venema and Tollervey, should provide greater mechanistic insight into the 1999) (Figure 1). Considering that this process is operational nature and biological function of the predicted to use 460% of resources in the cell (Warner, pathway, and more importantly, any contributions to human disease. For instance, why are there so many RPs that bind to and negatively regulate Mdm2? Do they have overlapping or redundant functions? Is the function of p53 in this context to suspend cell cycle progression until ribosome biogenesis is restored, or to remove individual cells from a population that are incapable of maintaining appropriate levels of protein synthesis?