Association of protein C23 with rapidly labeled nucleolar RNA

The association of nucleolar phosphoprotein C23 with preribosomal ribonucleoprotein (RNP) particles was examined in Novikoff hepatoma nucleoli. RNA was labeled with [3H]uridine for various times in cell suspensions, and RNP particles were extracted from isolated nucleoli and fractionated by sucrose gradient ultracentrifugation. The majority of protein C23 cosedimented with fractions containing rapidly labeled RNA (RL fraction). To determine whether there was a direct association of RNA with protein C23, the RL fraction was exposed to ultraviolet (UV) light (254 nm) for short periods of time. After 2 min of exposure there was a 50% decrease in C23 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, with no significant further decrease at longer times. When UV-treated fractions were subjected to phenol/chloroform extractions, as much as 30% of the labeled RNA was found in the phenol (protein) layer, indicating that RNA became cross-linked to protein. Similarly, there was an increase in protein C23 extracted into the water layer after irradiation. By SDS-PAGE analyses the cross-linked species migrated more slowly than protein C23, appearing as a smear detected either by [3H]uridine radioactivity or by anti-C23 antibody. With anti-C23 antibodies, up to 25% of the labeled RNA was precipitated from the RL fraction. Dot-blot hybridizations, using cloned rDNA fragments as probes, indicated that the RNA in the RL fraction and the immunoprecipitated RNA contained sequences from 18S and 28S ribosomal RNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of a new enzyme, NG,NG-dimethylarginine dimethylaminohydrolase, from rat kidney

A new enzyme, NG, NG-dimethylarginine dimethylaminohydrolase which plays a role in the metabolism of NG,NG-dimethyl-L-arginine, has been purified to homogeneity from rat kidney. The enzyme consists of a single polypeptide and its molecular weight is about 33,000. The isoelectric point of the enzyme is at pH 5.2. The enzyme catalyzes the hydrolytic liberation of the dimethylamino moiety of NG,NG-dimethyl-L-arginine and forms L-citrulline and dimethylamine. It is highly specific for NG,NG-dimethyl-L-arginine and NG-monomethyl-L-arginine, and the Km values for these amino acids are 0.18 and 0.36 mM, respectively. The enzyme shows the maximum activity at pH 6.5 and requires no cofactor. The activity is strongly inhibited by SH-blocking reagents (e.g. p-chloromercuribenzoate and HgCl2) and divalent metal ions (e.g. Cd2+, Cu2+, and Zn2+).     

Localization of nucleolar phosphoproteins B23 and C23 during mitosis

Nucleolar phosphoproteins B23 and C23 were simultaneously localized in unsynchronized male rat-kangaroo PtK2 cells during mitosis using a mouse monoclonal antibody against protein B23 and a rabbit antibody against protein C23. The distribution of proteins B23 and C23 during mitosis was compared with the distribution of the silver staining protein. During interphase, proteins B23 and C23 were both localized to the nucleolus. As the nucleolus disappeared in prophase, the distribution of protein B23 became nucleoplasmic, whereas most of protein C23 remained associated with the disappearing nucleolus. Throughout metaphase and anaphase protein B23 was found associated with the chromosomes, whereas protein C23 seemed to disappear. When the nucleolus reformed during telophase, protein C23 appeared first in ‘prenucleolar bodies’ and then in the nucleolus, whereas protein B23 did not appear in the nucleolus until late telophase or early G1 phase. Silver staining during mitosis closely paralleled the distribution of protein C23, supporting previous conclusions that protein C23 is a silver staining nucleolus organizer region (NOR) protein [19, 20].     

Silver staining,immunofluorescence, and immunoelectron microscopic localization of nucleolar phosphoproteins B23 and C23

Nucleolar organizer region (NOR)-specific silver staining and immunolocalization of nucleolar phosphoproteins B23 and C23 were compared in Novikoff hepatoma ascites cells. Silver staining and protein C23 immunostaining were both localized in the fibrillar shell surrounding the fibrillar center and in the fibrillar center. During mitosis, silver staining and protein C23 were localized at the NORs. Therefore, protein C23 and the silver-staining protein both seem to be associated with rDNA-containing structures (Mirre and Stahl 1981). A comparison of toluidine blue staining specific for RNA and B23 immunostaining demonstrated that protein B23 was associated with RNA-containing regions of the nucleolus and was absent from the fibrillar centers. Localization of these proteins and their functions are discussed in relation to the organization of the nucleolus.     

The ribonuclease activity of nucleolar protein B23.

Protein B23 is an abundant nucleolar protein and putative ribosome assembly factor. The protein was analyzed for ribonuclease activity using RNA-embedded gels and perchloric acid precipitation assays. Three purified bacterially expressed forms of the protein, B23.1, B23.2 and an N-terminal polyhistidine tagged B23.1 as well as the natural protein were found to have ribonuclease activity. However, the specific activity of recombinant B23.1 was approximately 5-fold greater than that of recombinant B23.2. The activity was insensitive to human placental ribonuclease inhibitor, but was inhibited by calf thymus DNA in a dose dependent manner. The enzyme exhibited activity over a broad range of pH with an apparent optimum at pH 7.5. The activity was stimulated by but not dependent on the presence of low concentrations of Ca2+, Mg2+ or NaCl. The Ca2+ effect was saturable and only stimulatory in nature. In contrast, Mg2+ and NaCl exhibited optimal concentrations for stimulation and both inhibited the ribonuclease at concentrations above these optima. These data suggest that protein B23 has intrinsic ribonuclease activity. The location of protein B23 in subcompartments of the nucleolus that contain preribosomal RNA suggests that its ribonuclease activity plays a role in the processing of preribosomal RNA.     

Mapping the functional domains of nucleolar protein B23

Protein B23 is a multifunctional nucleolar protein whose cellular location and characteristics strongly suggest that it is a ribosome assembly factor. The protein has nucleic acid binding, ribonuclease, and molecular chaperone activities. To determine the contributions of unique polypeptide segments enriched in certain classes of amino acid residues to the respective activities, several constructs that produced N- and C-terminal deletion mutant proteins were prepared. The C-terminal quarter of the protein was shown to be necessary and sufficient for nucleic acid binding. Basic and aromatic segments at the N- and C-terminal ends, respectively, of the nucleic acid binding region were required for activity. The molecular chaperone activity was contained in the N-terminal half of the molecule, with important contributions from both nonpolar and acidic regions. The chaperone activity also correlated with the ability of the protein to form oligomers. The central portion of the molecule was required for ribonuclease activity and possibly contains the catalytic site; this region overlapped with the chaperone-containing segment of the molecule. The C-terminal, nucleic acid-binding region enhanced the ribonuclease activity but was not essential for it. These data suggest that the three activities reside in mainly separate but partially overlapping segments of the polypeptide chain.     

Proteins C23 and B23 are the major nucleolar silver staining proteins.

To examine the silver staining proteins of Novikoff hepatoma nucleoli, the nucleolar proteins were separated on two-dimensional polyacrylamide gels with an isoelectric focusing first dimension and an acid-urea gel second dimension. The nucleoli were sequentially extracted with (1) 0.6 M potassium acetate, pH 5.5 and (2) 2 M potassium acetate — 5 M urea — 10 mM Tris, pH 7.5. The silver staining method used for the detection of silver binding proteins in gels was similar to that used to stain the nucleolar granules on microscope slides. Two major silver staining proteins were found which were identified as (molecular weight × 10^?3/pI) proteins C23 (100/5.3) and B23 (37/5.1). These two proteins are the major acidic proteins in Novikoff hepatoma nucleoli.     

Amino acid sequence and sites of phosphorylation in a highly acidic region of nucleolar nonhistone protein C23.

Novikoff hepatoma nucleolar nonhistone proteins, C23 and B23, contain highly acidic phosphorylated regions (Mamrack, M. D., et al. (1977) Biochem. Biophys. Res. Commun. 76, 150--157). Tryptic peptides from protein C23 containing these regions were purified by DEAE-Sephadex columns and paper electrophoresis at pH 1.8. One of these, peptide C23-Ca, was sequenced by combined automated and conventional methods. The proposed amino acid sequence is shown in eq 1. This peptide was found in three 32P-labeled forms with phosphoryl groups at positions 8 and 25, and probably 28. The highly acidic sequences adjacent to the phosphorylation sites represent a unique class of phosphorylation sites different from those in histones or substrates for cytoplasmic cAMP-dependent kinases. Ala-Ala-Pro-Ala-A5la-Pro-Ala-Ser-Glu-A10sp-Glu-Asp-Glu-Glu-A15sp-Asp-Asp-Asp-Glu-A20sp-Asp-Asp-Asp-Asp-S25er-Gln-Glu-Ser-Glu-G30lu-Glu-Asp-Glu-Glu-V35al-Met-Glu-Ile-Thr-P40ro-Ala-Lys (1).     

cDNA and deduced primary structure of rat protein B23, a nucleolar protein containing highly conserved sequences

Protein B23 (Mr/pI = 38,000/5.1) is a major RNA-associated nucleolar phosphoprotein which contains highly acidic segments and has a high affinity for silver ions. Using synthetic oligonucleotides as probes cloned cDNAs encoding protein B23 were isolated and characterized. One of the cDNAs, obtained from a rat brain library, contained an insert of 1232 base pairs of DNA encoding a polypeptide of 292 amino acid residues. Segments of the protein sequence were confirmed by partial sequencing of CNBr fragments from rat hepatoma protein B23. The protein contains a methionine-rich amino-terminal sequence and two highly acidic segments in the center of the sequence. The first acidic segment, in which 11 of the 13 residues are acidic, begins at residue 120 and contains a major phosphorylation site. In the second segment (residues 159-187) there are four copies of the sequence Asp-Asp-Glu, and all but two of the 29 residues have acidic side chains. When the sequence of the rat protein was compared with available sequences from other species a high degree of conservation was found; the 77-residue carboxyl-terminal sequence is identical with that of human protein B23 (Chan, P. K., Chan, W.-Y., Yung, B. Y. M., Cook, R. G., Aldrich, M. B., Ku, D., Goldknopf, I. L., and Busch, H. (1986) J. Biol. Chem. 261, 14335-24341), and about 63% of the residues are identical when the rat B23 sequence is compared with protein N038 from Xenopus laevis (Schmidt-Zachmann, M. S., Hügle-D?rr, B., and Franke, W. (1987) EMBO J. 6, 1881-1890). Except for the presence of highly acidic regions no significant similarities were found with protein C23 (nucleolin), the other major nucleolar protein.     

Synthesis by an improved solid-phase method of a highly acidic peptide from nucleolar nonhistone protein C23

The total synthesis of a 42-amino acid residue peptide corresponding to the proposed sequence of a highly acidic fragment of nucleolar nonhistone protein C23 was accomplished by an improved, mild solid-phase method, employing a p-alkoxybenzyl alcohol polystyrene resin. The symmetrical anhydride and active ester coupling were used exclusively. Coupling monitoring and amino acid analyses were carried out during the stepwise synthesis. The synthetic product was purified by gel filtration and ion-exchange chromatography, and found to be homogeneous by seven additional criteria. Phosphorylation of serine residues was attempted enzymatically, and the phosphopeptides obtained had electrophoretic mobilities comparable to that of the natural product.     

Identification of NG, NG-dimethylarginine in a nuclear protein from the lower eukaryote physarum polycephalum homologous to the major proteins of mammalian 40S ribonucleoprotein particles

A nuclear protein apparently homologous to the two major proteins of 40S heterogeneous nuclear ribonucleoprotein particles from mammalian cells has been isolated from the lower eukaryote $\text{Physarum polycephalum}$, purified, and found to contain a substantial amount of the unusual amino acid N^G, N^G-dimethylarginine. The apparent homology is based on similar molecular weights, basic isoelectric points and amino acid compositions including the dimethylarginine and a high content of glycine. The implications of the presence of this protein in $\text{Physarum polycephalum}$ and the possible significance of the N^G, N^G-dimethylarginine are discussed.     

The nucleolar protein, B-36, contains a glycine and dimethylarginine-rich sequence conserved in several other nuclear RNA-binding proteins

The amino terminal sequence of the 34 kD nucleolar protein B-36 isolated from the slime mold Physarum polycephalum has been determined. This portion of B-36 is rich in glycine, phenylalanine and the modified amino acid asymmetrical dimethylarginine (DMA) and is 65% identical to that for fibrillarin, a similar and potentially homologous 34 kD nucleolar protein from rat. The terminus of B-36 contains an interesting nine amino acid sequence, Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA-Gly, which is precisely repeated three times in the 110 kD nucleolar protein nucleolin. Similar sequences have also been reported in a yeast nucleolar protein (SSB-1) and several hnRNP proteins (rat A1 and brine shrimp GRP33). The conserved nature of this unusual sequence is suggestive of an important function which may include RNA-binding since several of these proteins share this feature.     

Function of nucleophosmin/B23, a nucleolar acidic protein, as a histone chaperone

We previously identified and purified a nucleolar phosphoprotein, nucleophosmin/B23, as a stimulatory factor for replication from the adenovirus chromatin. We show here that nucleophosmin/B23 functions as a histone chaperone protein such as nucleoplasmin, TAF-I, and NAP-I. Nucleophosmin/B23 was shown to bind to histones, preferentially to histone H3, to mediate formation of nucleosome, and to decondense sperm chromatin. These activities of B23 were dependent on its acidic regions as other histone chaperones, suggesting that B23/nucleophosmin is a member of histone chaperone proteins.     

Tumor suppressor ARF degrades B23, a nucleolar protein involved in ribosome biogenesis and cell proliferation

The tumor suppressor ARF induces a p53-dependent and -independent cell cycle arrest. Unlike the nucleoplasmic MDM2 and p53, ARF localizes in the nucleolus. The role of ARF in the nucleolus, the molecular target, and the mechanism of its p53-independent function remains unclear. Here we show that ARF interacts with B23, a multifunctional nucleolar protein involved in ribosome biogenesis, and promotes its polyubiquitination and degradation. Overexpression of B23 induces a cell cycle arrest in normal fibroblasts, whereas in cells lacking p53 it promotes S phase entry. Conversely, knocking down B23 inhibits the processing of preribosomal RNA and induces cell death. Further, oncogenic Ras induces B23 only in ARF null cells, but not in cells that retain wild-type ARF. Together, our results reveal a molecular mechanism of ARF in regulating ribosome biogenesis and cell proliferation via inhibiting B23, and suggest a nucleolar role of ARF in surveillance of oncogenic insults.     

The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins

Summary— Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at O°C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37°C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37°C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37°C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37°C in vitro, unlike to that what previous reports have indicated.     

Epitope distribution and immunochemical characterization of nucleolar phosphoprotein C23 using ten monoclonal antibodies

Summary Extractable nucleolar proteins from HeLa cells were used as a source of antigen to immunize mice for monoclonal antibody (MAb) production. Ten of the resulting MAbs shown to identify nucleolar phosphoprotein (110 kD/pI 5.5) were purified and used in immunochemical studies to further characterize protein C23. All ten MAbs showed nucleolar localization by indirect immunofluorescence; one antibody (FR2) also showed some nucleoplasmic localization that was attributed to a shared epitope between protein C23 and a 72 kD nuclear/nucleolar antigen. Reciprocal antibody cross blocking studies indicated that the ten MAbs identified nine distinct epitopes on protein C23. Interestingly, seven of the nine epitopes were shown by immunofluorescence and competitive ELISA studies to be species related. Immunostained patterns of exponentially growing HeLa cells suggest that protein C23 exists in vivo solely as a 110 kD peptide. However, protein C23 was subject to rapid degradation into a number of proteolytic fragments upon extraction or storage of isolated nucleoli. The failure to find protein C23 related peptides with molecular sizes less than 110 kD in exponentially growing cells and the lack of cytoplasmic localization of any of the ten MAbs suggests that protein C23 is not a prepro-protein processed in vivo to form ribosomal proteins as previously suggested (1).     

Localization of phosphoprotein C23 to nucleolar structures and to the nucleolus organizer regions

After extraction of Novikoff hepatoma nucleoli with 4 M urea/3 M LiCl, phosphoprotein C23 was isolated by DEAE-cellulose and Bio-Rad AG3-X4A column chromatography. Immunization of rabbits with the highly purified protein C23 resulted in the production of a specific antibody as determined by Ouchterlony diffusion analysis. When the immunoperoxidase method was used to localize protein C23 in cells, it was found in ‘fibrillar centers’ (nucleolonemas) in nucleoli. Protein C23 was also demonstrated to be present on the nucleolus organizer regions (NORs) of metaphase chromosomes.