Ribosomal protein S9 is a novel B23/NPM-binding protein required for normal cell proliferation

B23 (NPM/nucleophosmin) is a multifunctional nucleolar protein and a member of the nucleoplasmin superfamily of acidic histone chaperones. B23 is essential for normal embryonic development and plays an important role in genomic stability, ribosome biogenesis, and anti-apoptotic signaling. Altered protein expression or genomic mutation of B23 is encountered in many different forms of cancer. Although described as multifunctional, a genuine molecular function of B23 is not fully understood. Here we show that B23 is associated with a protein complex consisting of ribosomal proteins and ribosome-associated RNA helicases. A novel, RNA-independent interaction between ribosomal protein S9 (RPS9) and B23 was further investigated. We found that S9 binding requires an intact B23 oligomerization domain. Depletion of S9 by small interfering RNA resulted in decreased protein synthesis and G(1) cell cycle arrest, in association with induction of p53 target genes. We determined that S9 is a short-lived protein in the absence of ribosome biogenesis, and proteasomal inhibition significantly increased S9 protein level. Overexpression of B23 facilitated nucleolar storage of S9, whereas knockdown of B23 led to diminished levels of nucleolar S9. Our results suggest that B23 selectively stores, and protects ribosomal protein S9 in nucleoli and therefore could facilitate ribosome biogenesis.     

The nucleolus： reviewing oldies to have new understandings

The nucleolus is the most prominent compartment in the nucleus and known as the site for ribosome biogenesis ineucaryotes.In contrast,there is no such equivalent structure for ribosome synthesis in procaryotes.This raises twoconcerns that how does the nucleolus evolve and that whether the nucleolus remains playing a single role in ribosomebiogenesis along the evolution.Increasing data support new nucleolus functions,including signal recognition particleassembly,small RNA modification,telomerase maturation,cell-cycle and aging control,and cell stress sensor.Multiplefunctions of the nucleolus possibly result from the plurifunctionality of nucleolar proteins,such as nucleolin and Nopp 140.Proteomic analyses of human and Arabidopsis nucleolus lead a remarkable progress in understanding the evolution andnew functions of nucleoli.In this review,we present a brief history of nucleolus research and new concepts and unre-solved questions.Also,we introduce hepatitis D virus for studying the communication between the nucleolus and othersubnuclear compartments,and Caenorhabditis elegans for the role of nucleolus in the development and the epistaticcontrol of nucleologenesis.     

Human RBM28 protein is a specific nucleolar component of the spliceosomal snRNPs

The biogenesis of spliceosomal small nuclear RNAs (snRNAs) involves organized translocations between the cytoplasm and certain nuclear domains, such as Cajal bodies and nucleoli. Here we identify human RBM28 protein as a novel snRNP component, based on affinity selection of U6 small nuclear ribonucleoprotein (snRNP). As shown by immunofluorescence, RBM28 is a nucleolar protein. Anti-RBM28 immunoprecipitation from HeLa cell lysates revealed that this protein specifically associates with U1, U2, U4, U5, and U6 snRNAs. Our data provide the first evidence that RBM28 is a common nucleolar component of the spliceosomal ribonucleoprotein complexes, possibly coordinating their transition through the nucleolus.     

The nucleolus: a model for the organization of nuclear functions

Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs (rRNAs) are synthesized, processed and assembled with ribosomal proteins. The size and organization of the nucleolus are directly related to ribosome production. The organization of the nucleolus reveals the functional compartmentation of the nucleolar machineries that depends on nucleolar activity. When this activity is blocked, disrupted or impossible, the nucleolar proteins have the capacity to interact independently of the processing activity. In addition, nucleoli are dynamic structures in which nucleolar proteins rapidly associate and dissociate with nucleolar components in continuous exchanges with the nucleoplasm. At the time of nucleolar assembly, the processing machineries are recruited in a regulated manner in time and space, controlled by different kinases and form intermediate structures, the prenucleolar bodies. The participation of stable pre-rRNAs in nucleolar assembly was demonstrated after mitosis and during development but this is an intriguing observation since the role of these pre-rRNAs is presently unknown. A brief report on the nucleolus and diseases is proposed as well as of nucleolar functions different from ribosome biogenesis.Robert Feulgen Lecture presented at the 48th Symposium of the Society for Histochemistry in Stresa, Lake Maggiore, Italy, 7–10 September 2006.     

Nucleocytoplasmic traffic of CPEB1 and accumulation in Crm1 nucleolar bodies

The translational regulator CPEB1 plays a major role in the control of maternal mRNA in oocytes, as well as of subsynaptic mRNAs in neurons. Although mainly cytoplasmic, we found that CPEB1 protein is continuously shuttling between nucleus and cytoplasm. Its export is controlled by two redundant NES motifs dependent on the nuclear export receptor Crm1. In the nucleus, CPEB1 accumulates in a few foci most often associated with nucleoli. These foci are different from previously identified nuclear bodies. They contain Crm1 and were called Crm1 nucleolar bodies (CNoBs). CNoBs depend on RNA polymerase I activity, indicating a role in ribosome biogenesis. However, although they form in the nucleolus, they never migrate to the nuclear envelope, precluding a role as a mediator for ribosome export. They could rather constitute a platform providing factors for ribosome assembly or export. The behavior of CPEB1 in CNoBs raises the possibility that it is involved in ribosome biogenesis.     

Targeting the nucleolus as a therapeutic strategy in human disease

The nucleolus is the site of ribosome biogenesis, one of the most resource-intensive processes in eukaryotic cells. Accordingly, nucleolar morphology and activity are highly responsive to growth signaling and nucleolar insults which are collectively included in the actively evolving concept of nucleolar stress. Importantly, nucleolar alterations are a prominent feature of multiple human pathologies, including cancer and neurodegeneration, as well as being associated with aging. The past decades have seen numerous attempts to isolate compounds targeting different facets of nucleolar activity. We provide an overview of therapeutic opportunities for targeting nucleoli in different pathologies and currently available therapies.     

Molecular characterization of a novel nucleolar protein,pNO40

We report the discovery and characterization of a novel nucleolar protein. This protein, referred to as pNO40 based on its molecular weight on SDS-PAGE, was identified through yeast two hybrid interaction screen of a human kidney cDNA library using pinin (pnn) protein as the bait. The deduced amino acids of pNO40 derived from cDNA cloning of diverse species display high conservation; 95% identify between human and mouse and 57.3% identity for human and zebrafish. Several distinct domains are discernable in the ORF of pNO40, including a ribosomal protein S1 RNA binding region, a CCHC type zinc finger, and clusters of basic amino acid representing potential nucleolar targeting signal. Immunostaining of endogenous or transfected pNO40 indicated that it is localized to nucleoli of diverse cultured cells, with some concentration in the granular component of nucleoli. Northern blot analysis demonstrated that pNO40 message is expressed ubiquitously across all tissues examined. Characterization of human and mouse pNO40 gene revealed that mouse gene spans 44 kb in length and contains 8 exons, while that of human is 68 kb in length and displays two isoforms generated by alternative splicing of the 5(')-untranslated region and differential usage of translation start site. Based on sequence features and its subcellular location, we predict that pNO40 is a novel nucleolar protein with function related to ribosome maturation and/or biogenesis.     

Nucleolar retention of a translational C/EBPα isoform stimulates rDNA transcription and cell size

The messenger RNA of the intronless CEBPA gene is translated into distinct protein isoforms through the usage of consecutive translation initiation sites. These translational isoforms have distinct functions in the regulation of differentiation and proliferation due to the presence of different N‐terminal sequences. Here, we describe the function of an N‐terminally extended protein isoform of CCAAT enhancer‐binding protein α (C/EBPα) that is translated from an alternative non‐AUG initiation codon. We show that a basic amino‐acid motif within its N‐terminus is required for nucleolar retention and for interaction with nucleophosmin (NPM). In the nucleoli, extended‐C/EBPα occupies the ribosomal DNA (rDNA) promoter and associates with the Pol I‐specific factors u pstream‐b inding f actor 1 (UBF‐1) and SL1 to stimulate rRNA synthesis. Furthermore, during differentiation of HL‐60 cells, endogenous expression of extended‐C/EBPα is lost concomitantly with nucleolar C/EBPα immunostaining probably reflecting the reduced requirement for ribosome biogenesis in differentiated cells. Finally, overexpression of extended‐C/EBPα induces an increase in cell size. Altogether, our results suggest that control of rRNA synthesis is a novel function of C/EBPα adding to its role as key regulator of cell growth and proliferation.     

The SUMO system controls nucleolar partitioning of a novel mammalian ribosome biogenesis complex

Ribosome biogenesis is a tightly controlled pathway that requires an intricate spatial and temporal interplay of protein networks. Most structural rRNA components are generated in the nucleolus and assembled into pre-ribosomal particles, which are transferred for further maturation to the nucleoplasm and cytoplasm. In metazoa, few regulatory components for these processes have been characterized. Previous work revealed a critical role for the SUMO-specific protease SENP3 in the nucleolar steps of ribosome biogenesis. We biochemically purified a SENP3-associated complex comprising PELP1, TEX10 and WDR18, and demonstrate that this complex is involved in maturation and nucleolar release of the large ribosomal subunit. We identified PELP1 and the PELP1-associated factor LAS1L as SENP3-sensitive targets of SUMO, and provide evidence that balanced SUMO conjugation/deconjugation determines the nucleolar partitioning of this complex. This defines the PELP1-TEX10-WDR18 complex as a regulator of ribosome biogenesis and suggests that its SUMO-controlled distribution coordinates the rate of ribosome formation. These findings contribute to the basic understanding of mammalian ribosome biogenesis and shed new light on the role of SUMO in this process.     

Nucleophosmin is a binding partner of nucleostemin in human osteosarcoma cells

Nucleostemin (NS) is expressed in the nucleoli of adult and embryonic stem cells and in many tumors and tumor-derived cell lines. In coimmunoprecipitation experiments, nucleostemin is recovered with the tumor suppressor p53, and more recently we have demonstrated that nucleostemin exerts its role in cell cycle progression via a p53-dependent pathway. Here, we report that in human osteosarcoma cells, nucleostemin interacts with nucleophosmin, a nucleolar protein believed to possess oncogenic potential. Nucleostemin (NS) and nucleophosmin (NPM) displayed an extremely high degree of colocalization in the granular component of the nucleolus during interphase, and both proteins associated with prenucleolar bodies in late mitosis before the reformation of nucleoli. Coimmunoprecipitation experiments revealed that NS and NPM co-reside in complexes, and yeast two-hybrid experiments confirmed that they are interactive proteins, revealing the NPM-interactive region to be the 46-amino acid N-terminal domain of NS. In bimolecular fluorescence complementation studies, bright nucleolar signals were observed, indicating that these two proteins directly interact in the nucleolus in vivo. These results support the notion that cell cycle regulatory proteins congress and interact in the nucleolus, adding to the emerging concept that this nuclear domain has functions beyond ribosome production.