scholarly journals Association of Nonribosomal Nucleolar Proteins in Ribonucleoprotein Complexes during Interphase and Mitosis

1999 ◽  
Vol 10 (1) ◽  
pp. 77-90 ◽  
Author(s):  
Serafı́n Piñol-Roma
Keyword(s):  
Ribosomal Proteins ◽  
Rna Binding ◽  
Rrna Synthesis ◽  
28S Rrna ◽  
Ribosomal Subunits ◽  
Interphase Cell ◽  
Ribonucleoprotein Complexes ◽  
Nucleolar Proteins ◽  
M Phase ◽  
Nucleolar Rna

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.

scholarly journals Insight into Mammalian Selenocysteine Insertion: Domain Structure and Ribosome Binding Properties of Sec Insertion Sequence Binding Protein 2

2001 ◽  
Vol 21 (5) ◽  
pp. 1491-1498 ◽  
Author(s):  
Paul R. Copeland ◽  
Vincent A. Stepanik ◽  
Donna M. Driscoll
Keyword(s):  
Ribosomal Proteins ◽  
Insertion Sequence ◽  
Rna Binding ◽  
Stop Codon ◽  
Binding Protein ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Functional Domain ◽  
28S Rrna ◽  
Rna Binding Domain

ABSTRACT The cotranslational incorporation of the unusual amino acid selenocysteine (Sec) into both prokaryotic and eukaryotic proteins requires the recoding of a UGA stop codon as one specific for Sec. The recognition of UGA as Sec in mammalian selenoproteins requires a Sec insertion sequence (SECIS) element in the 3′ untranslated region as well as the SECIS binding protein SBP2. Here we report a detailed analysis of SBP2 structure and function using truncation and site-directed mutagenesis. We have localized the RNA binding domain to a conserved region shared with several ribosomal proteins and eukaryotic translation termination release factor 1. We also identified a separate and novel functional domain N-terminal to the RNA binding domain which was required for Sec insertion but not for SECIS binding. Conversely, we showed that the RNA binding domain was necessary but not sufficient for Sec insertion and that the conserved glycine residue within this domain was required for SECIS binding. Using glycerol gradient sedimentation, we found that SBP2 was stably associated with the ribosomal fraction of cell lysates and that this interaction was not dependent on its SECIS binding activity. This interaction also occurred with purified components in vitro, and we present data which suggest that the SBP2-ribosome interaction occurs via 28S rRNA. SBP2 may, therefore, have a distinct function in selecting the ribosomes to be used for Sec insertion.

Diverse relationships between metal ions and the ribosome

2021 ◽  
Author(s):  
Genki Akanuma
Keyword(s):  
Gene Expression ◽  
Secondary Structure ◽  
Metal Ions ◽  
Structural Stability ◽  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Rna Binding ◽  
Cellular Homeostasis ◽  
Ribosomal Subunits ◽  
Translational Activity

Abstract The ribosome requires metal ions for structural stability and translational activity. These metal ions are important for stabilizing the secondary structure of ribosomal RNA, binding of ribosomal proteins to the ribosome, and for interaction of ribosomal subunits. In this review, various relationships between ribosomes and metal ions, especially Mg2+ and Zn2+, are presented. Mg2+ regulates gene expression by modulating the translational stability and synthesis of ribosomes, which in turn contribute to the cellular homeostasis of Mg2+. In addition, Mg2+ can partly complement the function of ribosomal proteins. Conversely, a reduction in the cellular concentration of Zn2+ induces replacement of ribosomal proteins, which mobilizes free-Zn2+ in the cell and represses translation activity. Evolutional relationships between these metal ions and the ribosome are also discussed.

Effects of adenovirus infection on rRNA synthesis and maturation in HeLa cells

1983 ◽  
Vol 3 (4) ◽  
pp. 662-671
Author(s):  
C L Castiglia ◽  
S J Flint
Keyword(s):  
Hela Cells ◽  
Ribosomal Proteins ◽  
18S Rrna ◽  
Adenovirus Type ◽  
Cellular Protein ◽  
Rrna Synthesis ◽  
28S Rrna ◽  
Adenovirus Infection ◽  
Infected Cells ◽  
Infectious Cycle

The production of cytoplasmic and nucleolar rRNA species was examined in HeLa cells infected with high multiplicities of adenovirus type 5. Both 28S and 18S rRNA newly synthesized in infected cells ceased to enter the cytoplasm as reported previously (N. Ledinko, Virology 49: 79-89, 1972; H. J. Raskas, D. C. Thomas, and M. Green, Virology 40: 893-902, 1970). However, the effects on 28S cytoplasmic rRNA were observed considerably earlier in the infectious cycle than those on 18S rRNA. The inhibition of cellular protein synthesis and of the appearance in the cytoplasm of labeled cellular mRNA sequences (G. A. Beltz and S. J. Flint, J. Mol. Biol. 131: 353-373, 1979) were also monitored in infected cultures. During the later periods of an infectious cycle, from 18 h after infection, nucleolar rRNA synthesis and processing and exit of 18S rRNA from the nucleus were inhibited, probably reflecting the failure of infected cells to synthesize normal quantities of ribosomal proteins. The earliest responses of cellular RNA metabolism to adenovirus infection were, however, the rapid and apparently coordinate reductions in the levels of newly synthesized 28S rRNA and cellular mRNA sequences entering the cytoplasm.

Novel intron-encoded small nucleolar RNAs with long sequence complementarities to mature rRNAs involved in ribosome biogenesis

1995 ◽  
Vol 73 (11-12) ◽  
pp. 835-843 ◽  
Author(s):  
Jean-Pierre Bachellerie ◽  
Monique Nicoloso ◽  
Liang-Hu Qu ◽  
Bernard Michot ◽  
Michèle Caizergues-Ferrer ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Structural Basis ◽  
28S Rrna ◽  
Cis Acting ◽  
Nucleolar Proteins ◽  
Genes Encoding ◽  
Mouse Cells ◽  
Nucleolar Rnas

Recently, several new snoRNAs encoded in introns of genes coding for ribosomal, ribosome-associated, or nucleolar proteins have been discovered. We are presently studying four of these intronic snoRNAs. Three of them, U20, U21, and U24, are closely related to each other on a structural basis. They are included in genes encoding nucleolin and ribosomal proteins L5 and L7a, respectively, in warm-blooded vertebrates. These three metabolically stable snoRNAs interact with nucleolar protein fibrillarin. In addition, they display common features that make them strikingly related to snoRNA U14. U14 contains two tracts of complementarity to 18S rRNA, which are required for the production of 18S rRNA. U20 displays a 21 nucleotide (nt) long complementarity to 18S rRNA. U21 contains a 13 nt complementarity to an invariant sequence in eukaryotic 28S rRNA. U24 has two separate 12 nt long complementarities to a highly conserved tract of 28S rRNA. Phylogenetic evidences support the fundamental importance of the pairings of these three snoRNAs to pre-rRNA, which could be involved in a control of pre-rRNA folding during preribosome assembly. By transfection of mouse cells, we have also analyzed the processing of U20 and found that the -cis acting signals for its processing from intronic RNA are restricted to the mature snoRNA sequence. Finally, we have documented changes of host genes for these three intronic snoRNAs during the evolution of eukaryotes.Key words: snoRNA, pre-rRNA, folding, genes, introns.

scholarly journals The NF45/NF90 Heterodimer Contributes to the Biogenesis of 60S Ribosomal Subunits and Influences Nucleolar Morphology

2015 ◽  
Vol 35 (20) ◽  
pp. 3491-3503 ◽  
Author(s):  
Franziska Wandrey ◽  
Christian Montellese ◽  
Krisztian Koos ◽  
Lukas Badertscher ◽  
Lukas Bammert ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Rna Binding ◽  
Kinase Inhibitor ◽  
Affinity Purification ◽  
Spherical Shape ◽  
28S Rrna ◽  
Ribosomal Subunits ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Binding Domains ◽  
P53 Response

The interleukin enhancer binding factors ILF2 (NF45) and ILF3 (NF90/NF110) have been implicated in various cellular pathways, such as transcription, microRNA (miRNA) processing, DNA repair, and translation, in mammalian cells. Using tandem affinity purification, we identified human NF45 and NF90 as components of precursors to 60S (pre-60S) ribosomal subunits. NF45 and NF90 are enriched in nucleoli and cosediment with pre-60S ribosomal particles in density gradient analysis. We show that association of the NF45/NF90 heterodimer with pre-60S ribosomal particles requires the double-stranded RNA binding domains of NF90, while depletion of NF45 and NF90 by RNA interference leads to a defect in 60S biogenesis. Nucleoli of cells depleted of NF45 and NF90 have altered morphology and display a characteristic spherical shape. These effects are not due to impaired rRNA transcription or processing of the precursors to 28S rRNA. Consistent with a role of the NF45/NF90 heterodimer in nucleolar steps of 60S subunit biogenesis, downregulation of NF45 and NF90 leads to a p53 response, accompanied by induction of the cyclin-dependent kinase inhibitor p21/CIP1, which can be counteracted by depletion of RPL11. Together, these data indicate that NF45 and NF90 are novel higher-eukaryote-specific factors required for the maturation of 60S ribosomal subunits.

scholarly journals Effects of adenovirus infection on rRNA synthesis and maturation in HeLa cells.

1983 ◽  
Vol 3 (4) ◽  
pp. 662-671 ◽  
Author(s):  
C L Castiglia ◽  
S J Flint
Keyword(s):  
Hela Cells ◽  
Ribosomal Proteins ◽  
18S Rrna ◽  
Adenovirus Type ◽  
Cellular Protein ◽  
Rrna Synthesis ◽  
28S Rrna ◽  
Adenovirus Infection ◽  
Infected Cells ◽  
Infectious Cycle

The production of cytoplasmic and nucleolar rRNA species was examined in HeLa cells infected with high multiplicities of adenovirus type 5. Both 28S and 18S rRNA newly synthesized in infected cells ceased to enter the cytoplasm as reported previously (N. Ledinko, Virology 49: 79-89, 1972; H. J. Raskas, D. C. Thomas, and M. Green, Virology 40: 893-902, 1970). However, the effects on 28S cytoplasmic rRNA were observed considerably earlier in the infectious cycle than those on 18S rRNA. The inhibition of cellular protein synthesis and of the appearance in the cytoplasm of labeled cellular mRNA sequences (G. A. Beltz and S. J. Flint, J. Mol. Biol. 131: 353-373, 1979) were also monitored in infected cultures. During the later periods of an infectious cycle, from 18 h after infection, nucleolar rRNA synthesis and processing and exit of 18S rRNA from the nucleus were inhibited, probably reflecting the failure of infected cells to synthesize normal quantities of ribosomal proteins. The earliest responses of cellular RNA metabolism to adenovirus infection were, however, the rapid and apparently coordinate reductions in the levels of newly synthesized 28S rRNA and cellular mRNA sequences entering the cytoplasm.

scholarly journals M Phase Phosphoprotein 10 Is a Human U3 Small Nucleolar Ribonucleoprotein Component

1998 ◽  
Vol 9 (2) ◽  
pp. 437-449 ◽  
Author(s):  
Joanne M. Westendorf ◽  
Konstantin N. Konstantinov ◽  
Steven Wormsley ◽  
Mei-Di Shu ◽  
Naoko Matsumoto-Taniura ◽  
...  
Keyword(s):  
Coiled Body ◽  
Cell Fractionation ◽  
Rrna Processing ◽  
Novel Proteins ◽  
Nucleolar Proteins ◽  
Coiled Bodies ◽  
Interphase Cells ◽  
M Phase ◽  
Almost All ◽  
Nucleolar Rna

We have previously developed a novel technique for isolation of cDNAs encoding M phase phosphoproteins (MPPs). In the work described herein, we further characterize MPP10, one of 10 novel proteins that we identified, with regard to its potential nucleolar function. We show that by cell fractionation, almost all MPP10 was found in isolated nucleoli. By immunofluorescence, MPP10 colocalized with nucleolar fibrillarin and other known nucleolar proteins in interphase cells but was not detected in the coiled bodies stained for either fibrillarin or p80 coilin, a protein found only in the coiled body. When nucleoli were separated into fibrillar and granular domains by treatment with actinomycin D, almost all the MPP10 was found in the fibrillar caps, which contain proteins involved in rRNA processing. In early to middle M phase of the cell cycle, MPP10 colocalized with fibrillarin to chromosome surfaces. At telophase, MPP10 was found in cellular structures that resembled nucleolus-derived bodies and prenucleolar bodies. Some of these bodies lacked fibrillarin, a previously described component of nucleolus-derived bodies and prenucleolar bodies, however, and the bulk of MPP10 arrived at the nucleolus later than fibrillarin. To further examine the properties of MPP10, we immunoprecipitated it from cell sonicates. The resulting precipitates contained U3 small nucleolar RNA (snoRNA) but no significant amounts of other box C/D snoRNAs. This association of MPP10 with U3 snoRNA was stable to 400 mM salt and suggested that MPP10 is a component of the human U3 small nucleolar ribonucleoprotein.

scholarly journals Mutations in nucleolar proteins lead to nucleolar accumulation of polyA+ RNA in Saccharomyces cerevisiae.

1995 ◽  
Vol 6 (9) ◽  
pp. 1103-1110 ◽  
Author(s):  
T Kadowaki ◽  
R Schneiter ◽  
M Hitomi ◽  
A M Tartakoff
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Nucleolar Protein ◽  
Rrna Synthesis ◽  
Ribosomal Subunits ◽  
Nucleolar Proteins ◽  
Synthesis And Processing ◽  
Polymerase I ◽  
Nucleolar Components

Synthesis of mRNA and rRNA occur in the chromatin-rich nucleoplasm and the nucleolus, respectively. Nevertheless, we here report that a Saccharomyces cerevisiae gene, MTR3, previously implicated in mRNA transport, codes for a novel essential 28-kDa nucleolar protein. Moreover, in mtr3-1 the accumulated polyA+ RNA actually colocalizes with nucleolar antigens, the nucleolus becomes somewhat disorganized, and rRNA synthesis and processing are inhibited. A strain with a ts conditional mutation in RNA polymerase I also shows nucleolar accumulation of polyA+ RNA, whereas strains with mutations in the nucleolar protein Nop1p do not. Thus, in several mutant backgrounds, when mRNA cannot be exported i concentrates in the nucleolus. mRNA may normally encounter nucleolar components before export and proteins such as Mtr3p may be critical for export of both mRNA and ribosomal subunits.

Localisation of RNAs and proteins in nucleolar precursor bodies of early mouse embryos

2017 ◽  
Vol 29 (3) ◽  
pp. 509 ◽  
Author(s):  
Elena Lavrentyeva ◽  
Kseniya Shishova ◽  
German Kagarlitsky ◽  
Olga Zatsepina
Keyword(s):  
Ribosomal Proteins ◽  
Rna Binding ◽  
Mammalian Species ◽  
In Situ Hybridisation ◽  
Mouse Embryos ◽  
28S Rrna ◽  
Fluorescence In Situ Hybridisation ◽  
Rdna Transcription ◽  
Early Mouse ◽  
Nucleolar Precursor Bodies

Early embryos of all mammalian species contain morphologically distinct but transcriptionally silent nucleoli called the nucleolar precursor bodies (NPBs), which, unlike normal nucleoli, have been poorly studied at the biochemical level. To bridge this gap, here we examined the occurrence of RNA and proteins in early mouse embryos with two fluorochromes – an RNA-binding dye pyronin Y (PY) and the protein-binding dye fluorescein-5′-isothiocyanate (FITC). The staining patterns of zygotic NPBs were then compared with those of nucleolus-like bodies (NLBs) in fully grown surrounded nucleolus (SN)-type oocytes, which are morphologically similar to NPBs. We show that both entities contain proteins, but unlike NLBs, NPBs are significantly impoverished for RNA. Detectable amounts of RNA appear on the NPB surface only after resumption of rDNA transcription and includes pre-rRNAs and 28S rRNA as evidenced by fluorescence in situ hybridisation with specific oligonucleotide probes. Immunocytochemical assays demonstrate that zygotic NPBs contain rRNA processing factors fibrillarin, nucleophosmin and nucleolin, while UBF (the RNA polymerase I transcription factor) and ribosomal proteins RPL26 and RPS10 are not detectable. Based on the results obtained and data in the contemporary literature, we suggest a scheme of NPB assembly and maturation to normal nucleoli that assumes utilisation of maternally derived nucleolar proteins but of nascent rRNAs.

scholarly journals Localization of ribosomal protein S1 in the granular component of the interphase nucleolus and its distribution during mitosis.

1985 ◽  
Vol 100 (3) ◽  
pp. 873-886 ◽  
Author(s):  
B Hügle ◽  
R Hazan ◽  
U Scheer ◽  
W W Franke
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Cross Reactivity ◽  
Rrna Synthesis ◽  
Granular Component ◽  
Ribosomal Protein S1 ◽  
Nucleolar Proteins ◽  
Nucleolar Material ◽  
Cytoplasmic Ribosomes

Using antibodies to various nucleolar and ribosomal proteins, we define, by immunolocalization in situ, the distribution of nucleolar proteins in the different morphological nucleolar subcompartments. In the present study we describe the nucleolar localization of a specific ribosomal protein (S1) by immunofluorescence and immunoelectron microscopy using a monoclonal antibody (RS1-105). In immunoblotting experiments, this antibody reacts specifically with the largest and most acidic protein of the small ribosomal subunit (S1) and shows wide interspecies cross-reactivity from amphibia to man. Beside its localization in cytoplasmic ribosomes, this protein is found to be specifically localized in the granular component of the nucleolus and in distinct granular aggregates scattered over the nucleoplasm. This indicates that ribosomal protein S1, in contrast to reports on other ribosomal proteins, is not bound to nascent pre-rRNA transcripts but attaches to preribosomes at later stages of rRNA processing and maturation. This protein is not detected in the residual nucleolar structures of cells inactive in rRNA synthesis such as amphibian and avian erythrocytes. During mitosis, the nucleolar material containing ribosomal protein S1 undergoes a remarkable transition and shows a distribution distinct from that of several other nucleolar proteins. In prophase, the nucleolus disintegrates and protein S1 appears in numerous small granules scattered throughout the prophase nucleus. During metaphase and anaphase, a considerable amount of this protein is found in association with the surfaces of all chromosomes and finely dispersed in the cell plasm. In telophase, protein S1-containing material reaccumulates in granular particles in the nucleoplasm of the newly formed nuclei and, finally, in the re-forming nucleoli. These observations indicate that the nucleolus-derived particles containing ribosomal protein S1 are different from cytoplasmic ribosomes and, in the living cell, are selectively recollected after mitosis into the newly formed nuclei and translocated into a specific nucleolar subcompartment, i.e., the granular component. The nucleolar location of ribosomal protein S1 and its rearrangement during mitosis is discussed in relation to the distribution of other nucleolar proteins.

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