Nucleolar RNA-binding Protein Research Articles

The human cellular protein NoL12 is a specific partner of the HIV-1 nucleocapsid protein NCp7.

The human immunodeficiency virus-1 (HIV-1) nucleocapsid protein (NCp7) is a nucleic acid chaperone protein with two highly conserved zinc fingers. To exert its key roles in the viral cycle, NCp7 interacts with several host proteins. Among them, the human NoL12 protein (hNoL12) was previously identified in genome wide screens as a potential partner of NCp7. hNoL12 is a highly conserved 25 kDa nucleolar RNA-binding protein implicated in the 5'end processing of ribosomal RNA in the nucleolus and thus in the assembly and maturation of ribosomes. In this work, we confirmed the NCp7/hNoL12 interaction in cells by Förster resonance energy transfer visualized by Fluorescence Lifetime Imaging Microscopy and co-immunoprecipitation. The interaction between NCp7 and hNoL12 was found to strongly depend on their both binding to RNA, as shown by the loss of interaction when the cell lysates were pretreated with RNase. Deletion mutants of hNoL12 were tested for their co-immunoprecipitation with NCp7, leading to the identification of the exonuclease domain of hNoL12 as the binding domain for NCp7. Finally, the interaction with hNoL12 was found to be specific of the mature NCp7 and to require NCp7 basic residues. IMPORTANCE HIV-1 mature nucleocapsid (NCp7) results from the maturation of the Gag precursor in the viral particle and is thus mostly abundant in the first phase of the infection which ends with the genomic viral DNA integration in the cell genome. Most if not all the nucleocapsid partners identified so far are not specific of the mature form. We described here the specific interaction in the nucleolus between NCp7 and the human nucleolar protein 12, a protein implicated in ribosomal RNA maturation and DNA damage response. This interaction takes place in the cell nucleolus, a subcellular compartment where NCp7 accumulates. The absence of binding between hNoL12 and Gag makes hNoL12 one of the few known specific cellular partners of NCp7.

Nuclear phase separation directs HOX transcription in acute myeloid leukemia.

Cancer requires transcriptional misregulation to preserve a stem cell-like state, as in acute myeloid leukemia (AML) through sustained activation of HOX genes. Most drivers of HOX in AML have established roles in transcriptional regulation in the nucleus. In contrast, the most common driver, mutant Nucleophosmin1 (NPM1c) is a cytoplasmic oncoprotein resulting from the de novo addition of a nuclear export sequence to the essential nucleolar RNA binding protein NPM1. Our lab and others have shown that NPM1c is necessary for HOX activation; however, the mechanism underlying nuclear activity by this predominantly cytoplasmic protein remains perplexing. We used high-resolution microscopy to investigate NPM1c's localization and role in HOX transcription. Unexpectedly, we observed a fraction (<10%) of NPM1c resides in unreported nuclear puncta or condensates with >100-fold higher enrichment than in the cytoplasm. Biophysical characterization reveals that NPM1c condensates are phase-separated. These condensates require a critical concentration of NPM1c to form and exhibit a concentration-dependent increase in size and number across multiple cell lines. Modulation of NPM1c concentration and subsequent condensate number dictated HOX mRNA levels and cell growth. These results are consistent with a role for NPM1c condensates in HOX misregulation. Further evaluation of a dozen truncations of NPM1c allowed us to unequivocally establish a link between NPM1c condensates, HOX activation, and preservation of the stem cell-like state. Moreover, we observed NUP98 and MLL—proteins found in leukemic oncofusions—enrich in NPM1c condensates, leading us to characterize the ability of numerous fusion oncoproteins to drive nuclear phase separation and activate HOX. Our results suggest that nuclear phase separation is a ubiquitous mechanism for HOX activation, providing a common therapeutic vulnerability across AML subtypes and possibly other cancers.

Linking molecular-level RNA-protein interactions with mesoscale properties of the nucleolus.

The nucleolus is the most prominent nuclear body, and represents a condensate comprising the machinery of ribosome biogenesis, including multi-step transcription, processing, and assembly of ribosomal RNA (rRNA). These intricate steps are organized in well-characterized nucleolar substructures, with a host of RNA and proteins components that have been described in detail. However, the role of specific molecular-level interactions, such as RNA-RNA and RNA-protein, in governing nucleolar morphology and its material properties is unknown. Towards addressing this problem, we have deployed a suite of chemical biology, quantitative cellular microscopy and in vitro biophysical methods. Specifically, we use chemical perturbations to selectively and transiently inhibit distinct steps of ribosome biogenesis in cells. We then visualize and classify changes in nucleolar morphology as well as profile thermodynamics of nucleolar RNA-binding proteins (RBPs) upon treating cells with these inhibitors. Preliminary results indicate that the RBP thermodynamic profiles and morphologies are dependent on the presence or absence of specific RNA and/or RBP species in the nucleolus, suggesting that select RBP-RNA interactions may differentially contribute to nucleolar structure and function. To test this hypothesis, we are investigating material properties, dynamics, and RNA structural landscapes of in vitro nucleolar condensates with varying RNA and RBP compositions. Given that aberrant ribosome production is a hallmark of cancer and other diseases, our findings on the molecular thermodynamics of the nucleolus will pave the way towards rational therapeutic targeting.

Abstract 476: A human scFv as a tool to understand the biogenesis of a subset of oncogenic microRNAs

Abstract Trans-acting factors such as RNA-binding proteins (RBPs) and microRNAs (miRNA) have recently been recognized as prime regulators of gene expression during tumorigenesis. Nucleolin (NCL) is the most abundant nucleolar RNA-binding protein, controlling critical cellular processes such as chromatin remodeling, ribosome biogenesis and miRNA biogenesis. In the cytoplasm, NCL regulates gene expression post-transcriptionally by controlling mRNA stability and/or translation via ARE- and miRNA-mediated pathways. In contrast, NCL at the cell surface plays pivotal roles in cancer progression and metastasis in many types of tumors. Therefore, surface-NCL is an appealing target for cancer therapy. Earlier our group described 4LB5, a human-derived single chain fragment variable (scFv) that specifically targets surface-NCL exerting cytotoxic effects, both in vitro and in vivo. We have identified the central RNA-binding domains (RBD1-4) of NCL as 4LB5-interacting sites. 4LB5-mediated blocking of NCL-RBDs significantly impairs the biogenesis of a subset of oncogenic miRNAs, such as miR-21, -221 and -222, and their binding to the microprocessor complex. The structural fold of all the 4 RBDs is highly conserved, but there are significant differences in their sequences. RBD1-2 have been well studied in their mechanism and capability to form high-affinity RNA-binding complexes, but RBD 3-4 have not been explored to the same extent. We hypothesize that consecutive or selective combinations of RBDs in NCL might have distinct roles in RNA biology. NCL RBD1-2 domains are well documented in the literature for their high affinity for mRNAs and rRNAs. However, RNA-specificity on NCL-RBDs has yet to be elucidated. In this study, we used protein-antibody and protein-RNA docking analyses to identify potential differential binding affinities between multiple NCL modules (RDB1-2, RBD3-4 and RBD1-4) and either 4LB5 or miRNAs. In silico results were also evaluated by multiple biochemical assays (ELISA, Surface Plasmon Resonance, Pull-Down and Electrophoretic Mobility Shift Assays) using NCL Modules, 4LB5 and RNA molecules. Finally, competition assays were performed to assess ability of our scFv to impair miRNA binding to NCL. Our results indicate the importance of the NCL region targeted by 4LB5 in microRNA maturation. Functional studies will help us to better elucidate the biologic role of NCL RBDs, and their involvement in cancer pathogenesis. Citation Format: Valerio Embrione, Michael Scarpati, Zachary D. Uzzel, Rahimah Ahmad, Fortuna A. De Sury, Tyler J. Sheetz, Vincenzo Coppola, Shaneen M. Singh, Anjana D. Saxena, Carlo M. Croce, Dario Palmieri. A human scFv as a tool to understand the biogenesis of a subset of oncogenic microRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 476.

Nucleolin Controls Ribosome Biogenesis through Its RNA-Binding Properties

IntroductionNucleolin (NCL) is a multifunctional, proliferation-associated factor that is overexpressed in many cancers and has already been demonstrated to play a profound role in leukemogenesis (Abdelmohsen and Gorospe, 2012; Shen et al., 2014). This can be linked to an increased synthesis of ribosomal RNA (rRNA). Thus, in leukemic cells, high expression levels of NCL contribute to malignant transformation through the increase of rRNA synthesis, which is required to sustain high levels of protein synthesis. Physiologically, NCL is a highly abundant, nucleolar RNA-binding protein that is implicated in the regulation of polymerase I transcription, post-transcriptional gene regulation, and plays a central role in ribosome biogenesis (Srivastava and Pollard, 1999). To further elucidate the exact role of NCL, this study focused on the characterization of the RNA-binding properties and protein-interactions of NCL in the context of ribosome biogenesis.MethodsIn order to identify transcriptome-wide binding sites and the cellular RNA targets of NCL, PAR-CLIP (photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation) and RIP-Seq (RNA immunoprecipitation sequencing) analyses were carried out in HEK 293 cells. PAR-CLIP is characterized by the incorporation of 4-thiouridine into newly transcribed RNA that causes a T to C conversion in the corresponding cDNA of crosslinked RNA (Hafner et al., 2010). The RNA-binding properties and the interaction of NCL with its identified RNA targets were elucidated by electrophoretic mobility shift assays, isothermal titration calorimetry and size-exclusion chromatography. To further define the role of NCL in ribosome biogenesis and the effect on precursor rRNA levels, siRNA mediated knockdown of NCL was employed followed by RNA sequencing. Furthermore, to characterize the interaction network of NCL on a proteome-wide level, mass-spectrometry was performed.ResultsThis study focuses on the characterization of the RNA-binding properties of NCL and provides the first PAR-CLIP data set of NCL and identifies small nucleolar RNAs (snoRNA) and precursor rRNA as main targets of NCL, both of which were further confirmed by RIP-Seq analysis. Binding sites of NCL were identified in the 5'ETS (external transcribed spacer), after the first cleavage site, in ITS1 and ITS2 (internal transcribed spacer) within the precursor rRNA, indicating that NCL might play a role in the early processing steps of ribosome biogenesis within the nucleolus. Biochemical and structural binding analyses reveal that NCL interacts along the complete precursor region and shows high binding affinity to G/C/U-rich repeat sequences, which is in agreement with the nucleotide composition of the primary rRNA transcript. Moreover, we propose that siRNA mediated knockdown of NCL inhibits polymerase I transcription, which is shown by decreased expression levels of the precursor rRNA transcript. On the proteome-wide level, mass-spectrometry analysis of NCL identified several interaction partners including block of proliferation 1 (BOP1), DEAD-box RNA helicase 18 (DDX18), and 5'-3' exoribonuclease 2 (XRN2) and numerous ribosomal proteins of the small and the large ribosomal subunits including RPS24, RPL11, RPL35A, and RPL36.ConclusionThis study provides evidence that NCL is highly associated with the process of ribosome biogenesis on the proteome- and transcriptome-wide level. Therefore, NCL might serve as a promising biochemical target in the context of increased ribosome biogenesis in cancer. DisclosuresNo relevant conflicts of interest to declare.

Abstract 488: High DKC1 expression supports neuroblastoma tumor cell proliferation and is strongly associated with poor patient outcomes

Abstract Dyskerin is a nucleolar RNA-binding protein that functions as a core component of the telomerase holoenzyme, as well as in ribonuclear protein complexes involved in ribosome biogenesis and RNA processing. This study investigates the clinical and functional relevance of dyskerin expression in the paediatric cancer neuroblastoma. Here it is shown, in two independent neuroblastoma patient cohorts, that high expression of the gene encoding dyskerin (DKC1) very strongly correlates with poor event-free and overall survival. The prognostic power of DKC1 expression was independent of the established indicators used in the management of this disease, namely diagnosis age &amp;gt; 1.5 yr, stage of disease and amplification of the MYCN oncogene (P&amp;lt;0.0001). Dyskerin mRNA and protein expression were also found to be elevated, relative to normal cells, across a panel of neuroblastoma cell lines. Chromatin immunoprecipitation showed that the DKC1 promoter region was directly targeted by both MycN and c-Myc in neuroblastoma cells. Depletion of dyskerin using RNAi severely impaired proliferation, inhibited anchorage independent growth and dramatically slowed tumour growth in a mouse model of neuroblastoma (P&amp;lt;0.01, Two-way Anova). In p53 competent cells, the proliferative arrest induced by dyskerin depletion was accompanied by an accumulation of p53 and p21cip1, accumulation in G1 phase of the cell cycle and cell death. However in the absence of p53 function, an alternate mechanism operated to impair proliferation when dyskerin was downregulated. The mechanism responsible for the proliferative arrest also appeared to be telomerase-independent, as it was not rescued by ectopic overexpression of telomerase. Taken together, these data identify DKC1 is a crucial MYC target gene in neuroblastoma, and show that neuroblastoma cells become addicted to high DKC1 expression. The results support further investigation of DKC1 as a therapeutic target in the treatment of refractory neuroblastoma. Citation Format: Rosemary O'Brien, Michelle F. Maritz, Cheng Fei Kong, Stefania Purgato, Bing Liu, Chen Yang, Amanda Russell, Jayne Murray, Claudia Flemming, Michelle Haber, Giovanni Perini, Murray D. Norris, Jamie I. Fletcher, Karen L. Mackenzie. High DKC1 expression supports neuroblastoma tumor cell proliferation and is strongly associated with poor patient outcomes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 488. doi:10.1158/1538-7445.AM2015-488

Acute dyskerin depletion triggers cellular senescence and renders osteosarcoma cells resistant to genotoxic stress-induced apoptosis

Dyskerin is a conserved, nucleolar RNA-binding protein implicated in an increasing array of fundamental cellular processes. Germline mutation in the dyskerin gene (DKC1) is the cause of X-linked dyskeratosis congenita (DC). Conversely, wild-type dyskerin is overexpressed in sporadic cancers, and high-levels may be associated with poor prognosis. It was previously reported that acute loss of dyskerin function via siRNA-mediated depletion slowed the proliferation of transformed cell lines. However, the mechanisms remained unclear. Using human U2OS osteosarcoma cells, we show that siRNA-mediated dyskerin depletion induced cellular senescence as evidenced by proliferative arrest, senescence-associated heterochromatinization and a senescence-associated molecular profile. Senescence can render cells resistant to apoptosis. Conversely, chromatin relaxation can reverse the repressive effects of senescence-associated heterochromatinization on apoptosis. To this end, genotoxic stress-induced apoptosis was suppressed in dyskerin-depleted cells. In contrast, agents that induce chromatin relaxation, including histone deacetylase inhibitors and the DNA intercalator chloroquine, sensitized dyskerin-depleted cells to apoptosis. Dyskerin is a core component of the telomerase complex and plays an important role in telomere homeostasis. Defective telomere maintenance resulting in premature senescence is thought to primarily underlie the pathogenesis of X-linked DC. Since U2OS cells are telomerase-negative, this leads us to conclude that loss of dyskerin function can also induce cellular senescence via mechanisms independent of telomere shortening.

Dyskerin Localizes to the Mitotic Apparatus and Is Required for Orderly Mitosis in Human Cells

Dyskerin is a highly conserved, nucleolar RNA-binding protein with established roles in small nuclear ribonucleoprotein biogenesis, telomerase and telomere maintenance and precursor rRNA processing. Telomerase is functional during S phase and the bulk of rRNA maturation occurs during G1 and S phases; both processes are inactivated during mitosis. Yet, we show that during the course of cell cycle progression, human dyskerin expression peaks during G2/M in parallel with the upregulation of pro-mitotic factors. Dyskerin redistributed from the nucleolus in interphase cells to the perichromosomal region during prometaphase, metaphase and anaphase. With continued anaphase progression, dyskerin also localized to the cytoplasm within the mid-pole region. Loss of dyskerin function via siRNA-mediated depletion promoted G2/M accumulation and this was accompanied by an increased mitotic index and activation of the spindle assembly checkpoint. Live cell imaging further revealed an array of mitotic defects including delayed prometaphase progression, a significantly increased incidence of multi-polar spindles, and anaphase bridges culminating in micronucleus formation. Together, these findings suggest that dyskerin is a highly dynamic protein throughout the cell cycle and increases the repertoire of fundamental cellular processes that are disrupted by absence of its normal function.

NIR is a nucleolar RNA-binding protein that is involved in cell proliferation

A novel inhibitor of histone acetyltransferase repressor (NIR) is a histone acetylation inhibitor that associates with p53, p63 and histone complexes. Staining with anti-NIR antibodies revealed that the protein is localized at the nucleo- lus. The nucleolar localization of NIR was lost upon RNase treatment, whereas its localization was unaffected by treatment with DNase. These results indicate that NIR directly or indirectly binds RNA in the nucleolus. The expression of NIR pro- tein was higher in cancer cell lines than in non-cancer cell lines. The proliferation rate of human embryonic kidney 293T cells was higher following the ectopic expression of NIR than following the ectopic expression of green fluorescent protein (GFP). In summary, NIR is an RNA-associated protein that is involved in the cell proliferation regulation.

The Trypanosoma brucei Life Cycle Switch TbPTP1 Is Structurally Conserved and Dephosphorylates the Nucleolar Protein NOPP44/46

Trypanosoma brucei adapts to changing environments as it cycles through arrested and proliferating stages in the human and tsetse fly hosts. Changes in protein tyrosine phosphorylation of several proteins, including NOPP44/46, accompany T. brucei development. Moreover, inactivation of T. brucei protein-tyrosine phosphatase 1 (TbPTP1) triggers differentiation of bloodstream stumpy forms into tsetse procyclic forms through unknown downstream effects. Here, we link these events by showing that NOPP44/46 is a major substrate of TbPTP1. TbPTP1 substrate-trapping mutants selectively enrich NOPP44/46 from procyclic stage cell lysates, and TbPTP1 efficiently and selectively dephosphorylates NOPP44/46 in vitro. To provide insights into the mechanism of NOPP44/46 recognition, we determined the crystal structure of TbPTP1. The TbPTP1 structure, the first of a kinetoplastid protein-tyrosine phosphatase (PTP), emphasizes the conservation of the PTP fold, extending to one of the most diverged eukaryotes. The structure reveals surfaces that may mediate substrate specificity and affords a template for the design of selective inhibitors to interfere with T. brucei transmission.

Molecular cloning and characterization of Nop25, a novel nucleolar RNA binding protein, highly conserved in vertebrate species

We report here the identification and characterization of a novel nucleolar RNA binding protein, referred to as Nop25 as based on its predicted molecular size and subcellular location. Nop25 homologues were widely discovered in diverse vertebrate species as hypothetical proteins, but not found in yeasts, plants and prokaryotic organisms. Nop25 was ubiquitously expressed in adult mouse organs and constitutively during mouse embryogenesis. Indirect immunofluorescence analysis with an anti-Nop25 antibody, as well as an experiment using a GFP-fused protein, demonstrated that Nop25 was localized in the nucleolus. Treatment of the cells with a low doses of actinomycin D caused Nop25 to translocate to the periphery of the nucleolus, suggesting that nucleolar localization of Nop25 is associated with rRNA transcription. Treatment of COS7 cells with RNase A resulted in a complete dissociation of Nop25 from the nucleolus, while in vitro binding assay demonstrated that Nop25 could bind directly to single-stranded nucleic acids. Further characterization of associated RNA molecules with Nop25 using immunoprecipitation experiment showed that Nop25 might bind to 28S rRNA. Studies on this novel nucleolar RNA binding protein may provide new information on the intricate nucleolar machinery as related to the transcription and processing of rRNA molecules and/or the subsequent assembly and maturation of ribosomes.

RBD-1, a nucleolar RNA-binding protein, is essential for Caenorhabditis elegans early development through 18S ribosomal RNA processing.

RBD-1 is the Caenorhabditis elegans homolog of Mrd1p, which was recently shown to be required for 18S ribosomal RNA (rRNA) processing in yeast. To gain insights into the relationship between ribosome biogenesis and the development of multicellular organisms, we examined the expression and function of RBD-1. Maternal RBD-1 in the fertilized egg disappears immediately after cleavage starts, whereas zygotic RBD-1 first appears in late embryos and is localized in the nucleolus in most cells, although zygotic transcription of pre-rRNA is known to be initiated as early as the one-cell stage. RNA interference of the rbd-1 gene severely inhibits the processing of 18S rRNA in association with various developmental abnormalities, indicating its essential role in pre-rRNA processing and development in C.elegans. These results provide evidence for the linkage between ribosome biogenesis and the control of development and imply unexpected uncoupling of transcription and processing of pre-rRNA in early C.elegans embryos.

Expression of the human homologue of the small nucleolar RNA-binding protein NHP2 gene during monocytic differentiation of U937 cells

The human homologue of yeast NHP2, which is known to be a core protein component of yeast H/ACA small nucleolar ribonucleoprotein particles (snoRNPs), was identified by ODD-PCR as a 313-bp cDNA fragment exhibiting a distinct decrease in its expression level during TPA-induced differentiation of promonocytic U937 into monocytes and macrophages. Sequentially, a full-length cDNA of human NHP2 was isolated and the nucleotide sequence was determined. The NHP2 gene has a predicted 153 amino acid open reading frame, encoding 17.2 kDa protein that shares 38.4% and 44.4% identity with Saccharomyces cerevisiae NHP2 and Schizosaccharomyces pombe nhp2+, respectively. The TPA-induced differentiation of U937 cells, which also resulted in growth arrest, abruptly down-regulated the expression of NHP2. Removal of TPA restored cell growth through the retrodifferentiation process and subsequent expression of NHP2. NHP2 mRNA was markedly expressed in most tumor cells including Jurkat, K562, HL-60, U937, and HeLa S3. In healthy human tissues, NHP2 mRNA was expressed at high levels in spleen, thymus, prostate, testis, ovary, small intestine, colon (mucosal lining), heart, brain, placenta, skeletal muscle, kidney and pancreas, and at low levels in liver, and very weakly in peripheral blood leukocyte and lung. NHP2 mRNA, undetectable in human peripheral T cells, was induced at a maximum level between late G 1 and S phase after polyclonal activation. The expression of NHP2 mRNA during the cell cycle progression of Jurkat T cells also reached a maximum between late G 1 and S phase. These results indicate that the human NHP2 gene may be regulated at the transcription level depending on tissue specificity and cellular proliferative status, and that the down-regulation of NHP2 expression during induced differentiation of U937 cells may result from the growth arrest accompanying the differentiation.

The σ 70-like Motif : A Eukaryotic RNA Binding Domain Unique to a Superfamily of Proteins Required for Ribosome Biogenesis

Little is understood about the role of nucleolar RNA binding proteins in ribosome biogenesis, although there is a clear need for them based on the strict folding requirements of the pre-rRNA. We have identified a superfamily of RNA binding proteins whose members are required for different stages of ribosome biogenesis. The Imp4 superfamily is composed of five individual families (Imp4, Rpf1, Rpf2, Brx1, and Ssf) that all possess the σ 70-like motif, a eukaryotic RNA binding domain with prokaryotic origins. The Imp4 superfamily members associate with RNAs that are consistent with their distinct roles in ribosome biogenesis and suggest the mechanisms by which they function.

Nucleolin stimulates viral internal ribosome entry site-mediated translation

Previous results from our laboratory have identified a small (60 nt) RNA from the yeast S. cerevisiae that specifically inhibits internal ribosome entry site (IRES)-mediated translation programmed by poliovirus (PV) and hepatitis C virus (HCV) 5′-untranslated region (5′UTR). The yeast inhibitor RNA (called IRNA) was found to efficiently compete with viral 5′UTR for binding of several cellular polypeptides that presumably play important roles in IRES-mediated translation. One such IRNA (and 5′UTR)-binding protein has previously been identified as the La autoantigen. In this report, we have identified a 110-kDa IRNA-binding protein (which also interacts with viral 5′UTR) as nucleolin, a nucleolar RNA binding protein that was previously shown to translocate into the cytoplasm following infection of cells with poliovirus. We demonstrate that nucleolin (called C23) stimulates viral IRES-mediated translation both in vitro and in vivo. We also show that nucleolin mutants containing the carboxy-terminal RNA binding domains but lacking the amino terminal domain inhibit IRES-mediated translation in vitro. The translation inhibitory activity of these mutants correlates with their ability to bind the 5′UTR sequence. These results suggest a role of nucleolin/C23 in viral IRES-mediated translation.

Association of nonribosomal nucleolar proteins in ribonucleoprotein complexes during interphase and mitosis.

rRNA precursors are bound throughout their length by specific proteins, as the pre-rRNAs emerge from the transcription machinery. The association of pre-rRNA with proteins as ribonucleoprotein (RNP) complexes persists during maturation of 18S, 5.8S, and 28S rRNA, and through assembly of ribosomal subunits in the nucleolus. Preribosomal RNP complexes contain, in addition to ribosomal proteins, an unknown number of nonribosomal nucleolar proteins, as well as small nucleolar RNA-ribonucleoproteins (sno-RNPs). This report describes the use of a specific, rapid, and mild immunopurification approach to isolate and analyze human RNP complexes that contain nonribosomal nucleolar proteins, as well as ribosomal proteins and rRNA. Complexes immunopurified with antibodies to nucleolin-a major nucleolar RNA-binding protein-contain several distinct specific polypeptides that include, in addition to nucleolin, the previously identified nucleolar proteins B23 and fibrillarin, proteins with electrophoretic mobilities characteristic of ribosomal proteins including ribosomal protein S6, and a number of additional unidentified proteins. The physical association of these proteins with one another is mediated largely by RNA, in that the complexes dissociate upon digestion with RNase. Complexes isolated from M-phase cells are similar in protein composition to those isolated from interphase cell nuclear extracts. Therefore, the predominant proteins that associate with nucleolin in interphase remain in RNP complexes during mitosis, despite the cessation of rRNA synthesis and processing in M-phase. In addition, precursor rRNA, as well as processed 18S and 28S rRNA and candidate rRNA processing intermediates, is found associated with the immunopurified complexes. The characteristics of the rRNP complexes described here, therefore, indicate that they represent bona fide precursors of mature cytoplasmic ribosomal subunits.

Two RNA-binding Domains Determine the RNA-binding Specificity of Nucleolin

Nucleolin is an abundant nucleolar RNA-binding protein that seems to be involved in many aspects of ribosome biogenesis. Nucleolin contains four copies of a consensus RNA-binding domain (CS-RBD) found in several other proteins. In vitro RNA-binding studies previously determined that nucleolin interacts specifically with a short RNA stem-loop structure. Taken individually, none of the four CS-RBDs interacts significantly with the RNA target, but a peptide that contains the first two adjacent CS-RBDs (R12) is sufficient to account for nucleolin RNA-binding specificity and affinity. The full integrity of these two domains is required, since N- or C-terminal deletion abolishes the specific interaction with the RNA. Mutation of conserved amino acids within the RNP-1 sequence of CS-RBD 1 or 2 drastically reduces the interaction with the RNA, whereas mutation of the analogous residues in CS-RBDs 3 and 4 has no effect in the context of the R1234G protein (which corresponds to the C-terminal end of nucleolin). Our results demonstrate that nucleolin RNA-binding specificity is the result of a cooperation between two CS-RBDs (RBDs 1 and 2) and also suggests a direct or indirect involvement of the RNP-1 consensus sequence of both CS-RBDs in the recognition of the RNA target.

A Major Tyrosine-phosphorylated Protein of Trypanosoma brucei Is a Nucleolar RNA-binding Protein

We have previously identified a set of tyrosine-phosphorylated proteins with apparent molecular masses of 44-46 kDa as some of the major tyrosine phosphorylated species in the protozoan parasite Trypanosoma brucei. We now show that these molecules, herein named Nopp44/46, are localized in the nucleolus. Using monoclonal antibodies, we have isolated Nopp44/46 cDNA clones from expression libraries. Sequence analysis reveals that the predicted amino acid sequence of the molecule is composed of an N-terminal unique region, an internal acidic region, and C-terminal repeat region. Analysis of the cDNA clones and genomic Southern analysis indicated that Nopp44/46 belongs to a multigene family in which different gene copies are very similar but vary in the number of repeats. Interestingly, the repetitive amino acid sequence motif contains multiple RGG (Arg-Gly-Gly) boxes characteristic of RNA-binding proteins. In vitro binding experiments demonstrated that Nopp44/46 is indeed capable of binding nucleic acids. Competition experiments with different RNA homopolymers demonstrated that Nopp44/46 preferentially binds to poly(U). These studies suggest that Nopp44/46 may play a role in RNA metabolism in trypanosomes and raise the possibility that tyrosine phosphorylation may regulate the process.

NAB2: a yeast nuclear polyadenylated RNA-binding protein essential for cell viability.

A variety of nuclear ribonucleoproteins are believed to associate directly with nascent RNA polymerase II transcripts and remain associated during subsequent nuclear RNA processing reactions, including pre-mRNA polyadenylation and splicing as well as nucleocytoplasmic mRNA transport. To investigate the functions of these proteins by using a combined biochemical and genetic approach, we have isolated nuclear polyadenylated RNA-binding (NAB) proteins from Saccharomyces cerevisiae. Living yeast cells were irradiated with UV light to covalently cross-link proteins intimately associated with RNA in vivo. Polyadenylated RNAs were then selectively purified, and the covalent RNA-protein complexes were used to elicit antibodies in mice. Both monoclonal and polyclonal antibodies which detect a variety of NAB proteins were prepared. Here we characterize one of these proteins, NAB2. NAB2 is one of the major proteins associated with nuclear polyadenylated RNA in vivo, as detected by UV light-induced cross-linking. Cellular immunofluorescence, using both monoclonal and polyclonal antibodies, demonstrates that the NAB2 protein is localized within the nucleus. The deduced primary structure of NAB2 indicates that it is composed of at least two distinct types of RNA-binding motifs: (i) an RGG box recently described in a variety of heterogeneous nuclear RNA-, pre-rRNA-, mRNA-, and small nucleolar RNA-binding proteins and (ii) CCCH motif repeats related to the zinc-binding motifs of the largest subunit of RNA polymerases I, II, and III. In vitro RNA homopolymer/single-stranded DNA binding studies indicate that although both the RGG box and CCCH motifs bind poly(G), poly(U), and single-stranded DNA, the CCCH motifs also bind to poly(A). NAB2 is located on chromosome VII within a cluster of ribonucleoprotein genes, and its expression is essential for cell growth.

Nucleolin promotes secondary structure in ribosomal RNA

The effect of nucleolin on the secondary structure of RNA was studied using circular dichroism (CD). Nucleolin caused decreases in the main positive bands and shifts to higher wavelengths in the CD spectra of synthetic polynucleotides such as poly(G) and poly(A) indicating helix destabilizing activity. In contrast, nucleolin effected increases in signal and shifts to lower wavelengths of the peaks of CD spectra of ribosomal RNA, suggesting enhancement of secondary structure. Another major nucleolar RNA binding protein, B23, had helix destabilizing activity but did not enhance RNA secondary structure. It is proposed that nucleolin promotes formation of secondary structure in preribosomal RNA during the early stages of ribosome biogenesis.