Preribosomal ribonucleoprotein particles are a major component of a nucleolar matrix fraction

Biochemical and morphological studies were performed on Novikoff hepatoma ascites cell nucleolar matrix fractions prepared by deoxyribonuclease I digestion and high-molarity salt extractions essentially according to a published method [Berezney, R., & Buchholz, L. A. (1981) Exp. Cell Res. 20, 4995-5002]. The nucleolar matrix fraction was enriched in polypeptides of molecular mass of 28, 37.5, 40, 70, 72, 110 (protein C23), and 160 kDa, compared to the nuclear fraction in which polypeptides of molecular mass of 31, 33.5, 43.5, 46, 50, 56, and 59 kDa were predominant. About one-fourth of the protein, half of the RNA, and less than 4% of the DNA originally present in the nucleoli remained in the matrix fraction. Addition of single agents such as ethylenediaminetetraacetic acid, ribonuclease A, or mercaptoethanol during preparation had no significant effect on the polypeptide composition of the nucleolar matrix fraction. However, the combination of mercaptoethanol and ribonuclease A caused most of the RNA and protein to be removed, including protein C23 and the 160-kDa polypeptide, with polypeptides in the range of Mr 30 000-50 000 remaining. Electron microscopy of nucleolar matrix fractions revealed the presence of particles similar in size to the granular elements of nucleoli. However, when ribonuclease A and mercaptoethanol were included in the procedure, only amorphous material remained. Many proteins of nucleolar preribosomal RNP particles were also associated with the nucleolar matrix fraction. RNA from the nucleolar matrix fraction was enriched in sequences from 18S and 28S ribosomal RNA. These results indicate that preribosomal RNP particles are major constituents of a nucleolar matrix fraction prepared by the deoxyribonuclease I-high-molarity salt method.     

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)     

Ribonucleoprotein organization of eukaryotic RNA. XXX. Evidence that U1 small nuclear RNA is a ribonucleoprotein when base-paired with pre-messenger RNA in vivo

U1 small nuclear RNA is thought to be involved in messenger RNA splicing by binding to complementary sequences in pre-mRNA. We have investigated intermolecular base-pairing between pre-mRNA (hnRNA) and U1 small nuclear RNA by psoralen crosslinking in situ, with emphasis on ribonucleoprotein structure. HeLa cells were pulse-labeled with [3H]uridine under conditions in which hnRNA is preferentially labeled. Isolated nuclei were treated with aminomethyltrioxsalen , which produces interstrand crosslinks at sites of base-pairing between hnRNA and U1 RNA. hnRNA-ribonucleoprotein (hnRNP) particles were isolated in sucrose gradients containing 50% formamide, to dissociate non-crosslinked U1 RNA, and then analyzed by immunoaffinity chromatography using a human autoantibody that is specific for the ribonucleoprotein form of U1 RNA (anti-U1 RNP). After psoralen crosslinking, pulse-labeled hnRNA in hnRNP particles reproducibly bound to anti-U1 RNP. The amount of hnRNA bound to anti-U1 RNP was reduced 80 to 85% when psoralen crosslinking of nuclei was omitted, or if the crosslinks between U1 RNA and hnRNA were photo-reversed prior to immunoaffinity chromatography. Analysis of the proteins bound to anti-U1 RNP after crosslink reversal revealed polypeptides having molecular weights similar to those previously described for U1 RNP. These proteins did not bind to control, non-immune human immunoglobulin G. These results indicate that the subset of nuclear U1 RNA that is base-paired with hnRNA at a given time in the cell is a ribonucleoprotein. This raises the possibility that these proteins, as well as U1 RNA itself, may participate in pre-mRNA splice site recognition by U1 RNP.     

Fractionation of nucleoli from auxin-treated soybean hypocotyl into nucleolar chromatin and preribosomal particles

Nucleoli from auxin-treated tissues (Glycine max L. var Wayne or Kaoshiung No. 3) were isolated and purified by Percoll density gradient centrifugation. There was a 2.1-fold increase in RNA and a 2.8-fold increase in protein after a 24-h auxin treatment per unit nucleolar DNA. More than 150 acid-soluble protein spots were associated with the auxin-treated nucleoli on two dimensional (2-D) gel electropherograms.

Nucleoli from auxin-treated tissue were fractionated by suspension in 20 millimolar dithiothreitol at room temperature for 20 minutes into two distinct fractions referred to as the nucleolar chromatin and preribosomal particle fractions. The DNA:RNA:protein ratio of the chromatin fraction was 1:2.5:14. Most of RNA polymerase 1 activity and nucleolar DNA recovered in this fraction. The acid-soluble proteins in the chromatin were resolved into 32 protein spots on 2-D gel electropherogram. The most abundant spots were identified as histones.

The nucleolar preribosomal particle fraction had a DNA:RNA:protein ratio of 1:24:102 and contained only trace amounts of RNA polymerase 1 activity and only 10 per cent of the nucleolar DNA. Acid-soluble proteins associated with these particles were resolved into 78 protein spots; 72 of these (acid-soluble) protein spots corresponded in 2-D gel electrophoresis to 80S cytoplasmic ribosomal proteins. Some 15 protein spots found in 80S ribosomal proteins were absent in the preribosomal particles. It seems reasonable, based on these data, that the enlargement of nucleoli after auxin treatment is primarily due to the large increase in ribosomal proteins and rRNA which accumulate and assemble in the nucleoli in the form of preribosomal particles.

     

Isolation and proteomic characterization of the major proteins of the nucleolin-binding ribonucleoprotein complexes

Nucleolin (NCL) is one of the most abundant nucleolar proteins of exponentially growing eukaryotic cells. It is known to interact only transiently with rRNA and preribosomal particles and not to be detectable in mature cytoplasmic ribosomes, and is believed to function as multi-protein complexes during ribosome biogenesis and maturation. However, those multiprotein complexes remain only partially characterized due to the difficulty of conventional protein analysis methods. Here we report isolation of NCL-binding protein complex and its proteomic characterization with the use of an analytical method based on matrix-assisted laser desorption/ionization-time of flight analysis coupled with searching peptide mass databases. The NCL-binding protein complex was isolated by immunoprecipitation with anti-Flag antibody from human kidney 293 cells that were transfected with the Flag-tagged NCL gene, and showed RNA integrity for holding their protein constituents. Interaction between NCL and its binding complex was disrupted by an RNA oligonucleotide with a NCL recognition element, indicating that NCL binds to the ribonucleoprotein (RNP) complex mainly through the sequence specific protein-RNA interaction. We confirmed that an RNA-binding domain of NCL alone was sufficient to hold the entire NCL-binding RNP complex, indicating the strict binding specificity of NCL to the isolated RNP complex in 293 cells. We identified forty ribosomal proteins from both the large and small subunits, and twenty nonribosomal proteins. These results together suggest that the isolated NCL-binding RNP complex is a preribosomal particle present in the nucleolus of 293 cells.     

Isolation and characterization of a human U3 small nucleolar RNA gene

U3 RNA is an abundant, capped, small nucleolar RNA, implicated in the processing of preribosomal RNA. In this study, a DNA clone coding for U3 RNA (clone U3-1) was isolated from a human genomic library and characterized. The DNA sequence was identical to that of human U3 RNA isolated from HeLa cells. The flanking regions showed homology to the enhancer, promoter, and 3'-processing signal found in U1 and U2 snRNA genes. Further, the recently identified "U3 box" (GATTGGCTGCN10TATGTTAATTATGG) of rat U3 genes (Stroke and Weiner, (1985) J. Mol. Biol. 184, 183-193), was also found in the human U3 gene. This gene was transcribed in Xenopus oocytes; it is the first cloned true human U3 gene.     

Detection of a nucleolar 7-2 ribonucleoprotein and a cytoplasmic 8-2 ribonucleoprotein with autoantibodies from patients with scleroderma

In studies on antinucleolar antibodies in sera from 24 patients with scleroderma, an autoimmune disease, one serum, designated "anti-To", contained antibodies against a nucleolar 7-2 ribonucleoprotein and a novel cytoplasmic 8-2 ribonucleoprotein. The 7-2 and 8-2 RNAs are distinct RNAs with a pppG terminus. They are partially conserved between rat and human species and are present in distinct ribonucleoprotein particles. Eight sera contained antibodies that precipitated particles containing nucleolar U3 RNA; these antibodies appear to be directed against preribosomal particles containing U3 ribonucleoprotein, rather than the U3 ribonucleoprotein particles alone. All these ribonucleoproteins required proteins for antigenicity. These antibodies will be of use in studies on the structure and function of these novel small ribonucleoproteins.     

Globin messenger sequences in nuclear ribonucleoprotein particles of avian erythroblasts

Nuclear ribonucleoprotein particles were isolated from chick erythroblast nuclei. The particles were found to sediment as heterogeneous material. The major fraction of the rapidly synthesized RNP sedimented at 30 S, whereas the nuclei were found to contain a major, apparently more stable, RNP component sedimenting at about 40 S. The RNA isolated from the RNP particles was assayed for globin messenger activity in a wheat germ cell-free system. RNP sedimenting at relatively low S values (approx. 15 S) as well as RNP-particles of larger size code for globin. In addition to globin, the RNA of the particles codes also for other, not yet identified, proteins.     

RNA B is the major nucleolar trimethylguanosine-capped small nuclear RNA associated with fibrillarin and pre-rRNAs in Trypanosoma brucei.

RNA B is one of three abundant trimethylguanosine-capped U small nuclear RNAs (snRNAs) of Trypanosoma brucei which is not strongly identified with other U snRNAs by sequence homology. We show here that RNA B is a highly diverged U3 snRNA homolog likely involved in pre-rRNA processing. Sequence identity between RNA B and U3 snRNAs is limited; only two of four boxes of homology conserved between U3 snRNAs are obvious in RNA B. These are the box A homology, specific for U3 snRNAs, and the box C homology, common to nucleolar snRNAs and required for association with the nucleolar protein, fibrillarin. A 35-kDa T. brucei fibrillarin homolog was identified by using an anti-Physarum fibrillarin monoclonal antibody. RNA B and fibrillarin were localized in nucleolar fractions of the nucleus which contained pre-rRNAs and did not contain nucleoplasmic snRNAs. Fibrillarin and RNA B were precipitated by scleroderma patient serum S4, which reacts with fibrillarins from diverse organisms; RNA B was the only trimethylguanosine-capped RNA precipitated. Furthermore, RNA B sedimented with pre-rRNAs in nondenaturing sucrose gradients, similarly to U3 and other nucleolar snRNAs, suggesting that RNA B is hydrogen bonded to rRNA intermediates and might be involved in their processing.     

The nucleolus: an old factory with unexpected capabilities

The function of the nucleolus as a factory for assembling ribosomal subunits is well established, but many unrelated activities have been discovered over the past decade. Our understanding of the dynamics of nucleolar structure and its reassembly at the end of mitosis has recently advanced and the small nucleolar RNAs have been shown to be major players in the processing and modification of preribosomal RNA. Unexpectedly, the nucleolus also seems to play a role in nuclear export, sequestering regulatory molecules, modifying small RNAs, assembling ribonucleoprotein (RNP) and controlling aging.     

Phosphorylation of proteins of ribosomes and nucleolar preribosomal particles from Novikoff hepatmoa ascites cells

After labeling for two hours in vivo with ³²P-labeled orthophosphate, proteins from cytoplasmic ribosomes and nucleolar preribosomal particles of Novikoff hepatoma ascites cells were analyzed by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Five proteins (B2, B3, B6, B32 and B35P) were phosphorylated in the ribosomes. Approximately 19 proteins were phosphorylated in the nucleolar preribosomal particles; although four of these were ribosomal proteins, they were different from the proteins labeled in the ribosomes. The 15 additional phosphorylated nucleolar preribosomal particle proteins were non-ribosomal. These results suggest that phosphorylation of proteins of the nucleolar preribosomal particles is independent of phosphorylation of the cytoplasmic ribosomal proteins and may be a part of the maturation process of preribosomal particles.     

Effects of 5-azacytidine on nucleolar RNA and the preribosomal particles in Novikoff hepatoma cells.

Examination of nucleolar RNA from cultured Novikoff hepatoma cells treated for 3 hr with 5 x 10-4 M 5-azacytidine shows that significant amounts of analog-substituted 45S RNA are processed to the 32S RNA species, but 28S RNA formation is completely inhibited. Under these conditions of analog treatment 37% of the cytidine residues in the 45S RNA is replaced by 5-azacytidine. During coelectrophoresis of nucleolar RNA from 5-azacytidine-treated and control cells, the analog-substituted 45S RNA and 32S RNA display reduced mobilities compared to the control 45S RNA and 32S RNA. Coelectrophoresis of analog-substituted and control RNA after formaldehyde denaturation shows no differences in electrophoretic mobility between the two RNA samples, suggesting that 5-azacytidine incorporation may alter the secondary structure of the 45S RNA and the 32S RNA. 5-Azacytidine at 5 x 10-4 M severely inhibits protein synthesis in Novikoff cells by 3 hr. After this length of treatment, however, CsCl buoyant density analysis reveals no difference in density of either the 80S or 55S preribosomal ribonucleoprotein particles when compared to normal particles. Also 5-azacytidine treatment does not appear to cause major changes in the polyacrylamide gel electrophoresis patterns of the proteins in the 80S and 55S preribosomal particles. These results together with previous findings suggest that 5-azacytidine's inhibition of rRNA processing is possibly related to its alteration of the structure of the ribosomal precursor RNAs and is not a consequence of a general inhibition of ribosomal protein formation.     

RNA binding fragments from nucleolin contain the ribonucleoprotein consensus sequence

Nucleolin (C23 or 100 kDa) is a major nucleolar phosphoprotein whose primary structure has recently been determined (Lapeyre, B., Bourbon, H., and Amalric, F. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1472-1476) and found to be associated with preribosomal RNA (Herrera, A. H., and Olson, M. O. J. (1986) Biochemistry 25, 6258-6263). To identify the RNA binding region of the molecule, cyanogen bromide fragments were tested for binding of 18 S and 28 S ribosomal RNA by a "Western blotting" technique. Fragments with apparent molecular masses of 13, 33, and 47 kDa bound RNA with no preference for either 18 S or 28 S RNA. By protein sequencing, these fragments were localized in the carboxyl-terminal two-thirds of the molecule. The nucleolin sequence was searched for the ribonucleoprotein consensus sequence found in other RNA binding proteins. Four copies of a closely related 11-residue sequence were found within 80-90 residue repeats in the RNA binding region between residues 285 and 629. These results suggest that a highly conserved structure for the binding of different classes of RNA is utilized by several proteins.     

Murine graft vs host disease. A model for study of mechanisms that generate autoantibodies to ribonucleoproteins

We established chronic graft vs host disease in (BALB/c x A/J) F1 mice with the injection of lymphoid cells from the parental A/J strain. These animals developed glomerulonephritis, forefoot edema, alopecia, splenomegaly, and lymphadenopathy to various degrees, and all developed antinuclear antibodies. To determine whether these antibodies were directed against the small nuclear ribonucleoprotein (snRNP) particles that are characteristic targets for autoimmune responses in human rheumatic diseases, sera were studied in the 32P immunoprecipitation and immunoblotting assays. Among 20 mice, antibodies to snRNP developed in 10. These antibodies usually reached maximal levels about 4 wk after induction of graft vs host disease and generally fell thereafter. However, two mice developed antibodies to snRNP between the 10th and 20th wk of follow-up. Sera from six mice strongly recognized the U1 snRNP and an additional serum strongly bound both the U1 and U3 particles. Several sera contained lower levels of antibodies specific for the U3 and possibly pre-U2 snRNP particles. In immunoblots, sera that immunoprecipitated the U1 snRNP bound epitopes located on its 70,000 Da, A, B'/B, and/or C polypeptides. Sera that immunoprecipitated the U3 snRNP recognized a 34,000-Da polypeptide. These polypeptides are known to bear the autoantigenic epitopes that are recognized by human sera containing anti-U1 RNP and anti-U3 RNP autoantibodies. We conclude that chronic graft vs host disease in mice provides a model for the study of the autoimmune responses that characterize human diseases such as mixed connective tissue disease, scleroderma, and SLE.     

The translocation inhibitor tuberactinomycin binds to nucleic acids and blocks the in vitro assembly of 50S subunits.

Binding studies were performed with a [14C]-labelled derivative of viomycin, tuberactinomycin 0 (TUM O). TUM O bound to 30S and 50S subunits. The binding component was the RNA, since ribosomal proteins did not bind the drug. Other RNAs such as tRNA, phage RNA (MS2), and homopolynucleotides also bound the drug. Striking differences in the binding capacity of the various homopolynucleotides were found. Poly(U) bound strongly, poly(G) and poly(C) bound intermediately, whereas poly(A) showed a very low binding. DNA also bound TUM O, although with native DNA the binding was only weak. Finally the effects of viomycin on the assembly in vitro of the 50S subunit from E. coli were tested. A very strong inhibition was found: when the reconstitution was performed at 0.5 x 10(-6) M viomycin the particles formed sedimented at about 50S, but showed a residual activity of less than 10%. The inhibitory power of viomycin with respect to the in vitro assembly is more pronounced than that found in in vitro systems for protein synthesis.     

Studies on the Function of Intracellular Ribonucleases : II. The Interaction of Ribonucleoprotein and Enzymes

To determine the possible significance of in vivo or in vitro enzyme action in ribonucleoprotein systems, rat liver microsomes and ribonucleoprotein particles (RNP) prepared from them by deoxycholate treatment were incubated for 1 hour at 37°C. with crystalline pancreatic ribonuclease (RNase) or various RNase-free crystalline proteolytic enzymes. The extent of the degradation of the RNA of the microsomes and RNP was determined and the protein degradation estimated in both cases. With either microsomes or RNP, RNase (0.5 to 1.0 mg. per ml.) degraded from 75 to 95 per cent of the RNA, with little protein breakdown being apparent when microsomes were used but with significant protein degradation in the RNP. When microsomes were treated with proteolytic enzymes approximately 40 to 50 per cent of the original microsomal protein became nonsedimentable while at the same time 60 to 80 per cent of the RNA was also found to be non-sedimentable. Of the non-sedimentable RNA, approximately one-third was in the form of acid-precipitable RNA while the remainder was in the form of acid-soluble nucleotides. When RNP was treated with proteolytic enzymes, about 95 per cent of the RNA could no longer be sedimented. About half of this appeared as acid-precipitable RNA and half as acid-soluble nucleotides. Both microsomes and RNP contained significant RNase activity with RNP exhibiting about 10 times the specific activity of microsomes. Some of the characteristics of this RNase activity were determined and the results with proteolytic enzymes interpreted in light of this activity.     

Detection of beta-globin mRNA precursor in heterogeneous nuclear ribonucleoprotein associated with U1-RNP by using anti-RNP antibody

Heterogeneous nuclear RNA-ribonucleoprotein (hnRNP) fractions were isolated from Friend erythroleukemia cells and separated by 15-45% sucrose gradient centrifugation. The distribution of small nuclear RNAs (snRNAs) in hnRNP fractions indicated that the snRNAs are associated with hnRNP particles. HnRNP fractions were incubated with normal IgG or anti-U1 RNP IgG, and the resulting immunocomplexes were isolated by binding to a protein A-Sepharose column. HnRNP was found in bound fractions only when anti-U1 RNP IgG was used. By Northern hybridization of RNA extracted from the immunocomplexes with a beta-globin genomic DNA probe, 15S beta-globin mRNA precursors and 10S mature mRNA were detected. These findings suggest the existence of a complex of U1 RNP particles and hnRNP particles containing beta-globin pre-mRNA.