Ribocharin: a nuclear Mr 40,000 protein specific to precursor particles of the large ribosomal subunit

Hügle, Barbara; Scheer, Ulrich; Franke, Werner W.

doi:10.1016/s0092-8674(85)80034-9

Cited by 88 publications

(55 citation statements)

References 58 publications

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“…Recently, Hiigle et al [26] identified a nuclear/nucleolar Mr 40,000 protein in Xenopus laevis and other amphibia which appears to be specific to precursor particles of the large ribosomal subunit. This protein , which was named ribocharin, was specifically mapped to the granular component of the nucleolus and nucleoplasmic 65 S particles in amphibia.…”

Section: Discussionmentioning

confidence: 99%

Immunolocalization of 7-2-ribonucleoprotein in the granular component of the nucleolus

Reimer

Raška

Scheer

et al. 1988

Experimental Cell Research

108

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Certain autoimmune sera contain antibodies against a nucleolar ribonucleoprotein particle associated with 7-2-RNA (R. Reddy et al. (1983) J . Bioi. Chem . 258, 1383C. Hashimoto and J. A. Steitz (1983) J. Bioi. Chem. 258, 1379). In this study, we showed by immunofluorescence microscopy that antibodies reactive with 7-2-ribonucleoprotein immunolocalized in the granular regions of actinomycin D and 5,6-dichloro-I-j3-D-ribofuranosylbenzimidazole (DRB)-segregated nucleoli from Vero cells. By electron microscopic immunocytochemistry, antigen-antibody complexes were located in the granular component of transcriptionally active nucleoli from rat liver hepatocytes and HeLa cells. Anti-7-2-RNP antibodies from two autoimmune sera immunoprecipitated a major protein of Mr 40,000 from e 5 S] methionine-Iabeled HeLa cell extract. The immunolocalization data suggest that 7-2-ribonucleoprotein may be involved in stages of ribosome biogenesis which take place in the granular component of the nucleolus, i.e., assembly, maturation , and/or transport of preribosomes .

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Section: Discussionmentioning

confidence: 99%

Immunolocalization of 7-2-ribonucleoprotein in the granular component of the nucleolus

Reimer

Raška

Scheer

et al. 1988

Experimental Cell Research

108

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“…Because fractionation of isolated Xenopus oocyte nuclei by differential centrifugation leads to well-defined protein fractions characterized by specific nuclear components (cf. Hü gle et al, 1985;Kö hler et al, 1997; SchmidtZachmann et al, 1998), the distribution of protein NOH61 in such fractions from oocyte nuclei, egg extracts, and somatic cells was analyzed by immunoblotting with the NOH61-specific antibody (Figure 4). Antibody NOH61-5.1, which shows the strongest cross-reactivity with the Xenopus homo- logue, recognized its antigen in total oocyte nuclei as well as in the LSP and HSP fractions, indicating that protein NOH61 is a nuclear protein bound to relatively large structures, i.e., the nucleoli (LSP) and extranucleolar nucleoplasmic particles (HSP), respectively.…”

Section: Biochemical Characterization Of Protein Noh61mentioning

confidence: 99%

“…Subsequent fractionation of nuclear contents by differential centrifugation was performed as described in detail by Hü gle et al (1985), resulting in fractions termed "low-speed pellet" (LSP), "high-speed pellet" (HSP), and "high-speed supernatant" (HSS). The LSP fractions were cleared from yolk proteins by Freon extraction (Evans and Kay, 1991).…”

Section: Isolation and Fractionation Of Xenopus Oocyte Nuclei And Eggmentioning

confidence: 99%

“…The isolation of preribosomal particles as well as the preparation of ribosomes and ribosomal subunits have been described in detail before (Hü gle et al, 1985).…”

Section: Sucrose Gradient Density Centrifugation and Gel Filtrationmentioning

confidence: 99%

“…Schmidt-Zachmann et al, 1998). The detection of protein NOH61 in the HSP fraction was of special interest, because it has been shown that this fraction is highly enriched in free nucleoplasmic preribosomes (Hü gle et al, 1985). To examine whether protein NOH61 might be complexed with preribosomal particles, the HSP fraction was further separated by centrifugation in 10 -40% sucrose gradients, and the resulting fractions were analyzed by SDS-PAGE and immunoblotting ( Figure 5).…”

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confidence: 99%

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A Novel Helicase-Type Protein in the Nucleolus: Protein NOH61

Zirwes

Eilbracht

Kneissel

et al. 2000

MBoC

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We report the identification, cDNA cloning, and molecular characterization of a novel, constitutive nucleolar protein. The cDNA-deduced amino acid sequence of the human protein defines a polypeptide of a calculated mass of 61.5 kDa and an isoelectric point of 9.9. Inspection of the primary sequence disclosed that the protein is a member of the family of "DEAD-box" proteins, representing a subgroup of putative ATP-dependent RNA helicases. ATPase activity of the recombinant protein is evident and stimulated by a variety of polynucleotides tested. Immunolocalization studies revealed that protein NOH61 (nucleolar helicase of 61 kDa) is highly conserved during evolution and shows a strong accumulation in nucleoli. Biochemical experiments have shown that protein NOH61 synthesized in vitro sediments with ϳ11.5 S, i.e., apparently as homo-oligomeric structures. By contrast, sucrose gradient centrifugation analysis of cellular extracts obtained with buffers of elevated ionic strength (600 mM NaCl) revealed that the solubilized native protein sediments with ϳ4 S, suggestive of the monomeric form. Interestingly, protein NOH61 has also been identified as a specific constituent of free nucleoplasmic 65S preribosomal particles but is absent from cytoplasmic ribosomes. Treatment of cultured cells with 1) the transcription inhibitor actinomycin D and 2) RNase A results in a complete dissociation of NOH61 from nucleolar structures. The specific intracellular localization and its striking sequence homology to other known RNA helicases lead to the hypothesis that protein NOH61 might be involved in ribosome synthesis, most likely during the assembly process of the large (60S) ribosomal subunit. INTRODUCTIONNucleoli, the most conspicuous intranuclear structures in eukaryotic cells, are known to be the main site of ribosome biosynthesis (Hadjiolov, 1985;Scheer and Weisenberger, 1994;Scheer and Hock, 1999). More recently, however, it has also become clear that the nucleolus has additional functions and may be involved in the transport or assembly of various kinds of ribonucleoprotein particles (reviewed by Pederson, 1998).The production of preribosomal particles is a complex process, involving the transcription of the rRNA genes, the processing of the primary transcripts, and the addition of proteins to the nascent preribosomes as well as the incorporation of the 5S rRNA, which is synthesized outside of the nucleolus. Besides the rRNA gene clusters and flanking sequences, nucleoli contain a large number of proteins and RNA components that are not part of mature cytoplasmic ribosomes but are involved in the transcriptional (Reeder, 1990;Paule, 1993) and post-transcriptional regulation of ribosome synthesis (Olson, 1990). Among these are small nucleolar RNAs (snoRNAs), which play a major role in the endo-and exonucleolytic processing of the large preribosomal precursor as well as in the specific modification of rRNA molecules by remodeling certain uridine residues into pseudouridines and by tagging ribose moieties with methyl groups (To...

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Nucleolar targeting of 5S RNA inXenopus laevis oocytes: Somatic-type nucleotide substitutions enhance nucleolar localization

North¹,

Allison²

1998

J. Cell. Biochem.

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In Xenopus laevis oocytes, 5S RNA is stored in the cytoplasm until vitellogenesis, at which time it is imported into the nucleus and targeted to nucleoli for ribosome assembly. This article shows that throughout oogenesis there is a pool of nuclear 5S RNA which is not nucleolar-associated. This distribution reflects that of oocyte-type 5S RNA, which is the major 5S RNA species in oocytes; only small amounts of somatic-type, which differs by six nucleotides, are synthesized. Indeed, 32P-labeled oocyte-type 5S RNA showed a degree of nucleolar localization similar to endogenous 5S RNA (33%) after microinjection. In contrast, 32P-labeled somatic-type 5S RNA showed significantly enhanced localization, whereby 70% of nuclear RNA was associated with nucleoli. A chimeric RNA molecule containing only one somatic-specific nucleotide substitution also showed enhanced localization, in addition to other somatic-specific phenotypes, including enhanced nuclear import and ribosome incorporation. The distribution of 35S-labeled ribosomal protein L5 was similar to that of oocyte-type 5S RNA, even when preassembled with somatic-type 5S RNA. The distribution of a series of 5S RNA mutants was also analyzed. These mutants showed various degrees of localization, suggesting that the efficiency of nucleolar targeting can be influenced by many discrete regions of the 5S RNA molecule.

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Ribocharin: a nuclear Mr 40,000 protein specific to precursor particles of the large ribosomal subunit

Cited by 88 publications

References 58 publications

Immunolocalization of 7-2-ribonucleoprotein in the granular component of the nucleolus

Immunolocalization of 7-2-ribonucleoprotein in the granular component of the nucleolus

A Novel Helicase-Type Protein in the Nucleolus: Protein NOH61

Nucleolar targeting of 5S RNA inXenopus laevis oocytes: Somatic-type nucleotide substitutions enhance nucleolar localization

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