Human autoantibodies: probes for nucleolus structure and function

Reimer, Georg; Raška, Ivan; Tan, Eng M.; Scheer, Ulrich

doi:10.1007/bf02899205

Cited by 120 publications

(54 citation statements)

References 72 publications

(67 reference statements)

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“…This has been shown with antibodies reacting with various constituents of the dense fibrillar component as well as the granular component (e.g. Schmidt-Zachmann et al, 1984;Hiigle et al, 1985;Reimer et al, 1987a;Raska et al, 1989).…”

Section: Ill Immunolocaliza Non Of Histonesmentioning

confidence: 90%

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level

Thiry

Scheer

Goessens

1991

Electron Microscopy Reviews

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Abstract-Nucleoli are the morphological expression of the activity of a defined set of chromosomal segments bearing rRNA genes. The topological distribution and composition of the intranucleolar chromatin as well as the definition of nucleolar structures in which enzymes of the rDNA transcription machinery reside have been investigated in mammalian cells by various immunogold labelling approaches at the ultrastructural level. The precise intranucleolar location of rRNA genes has been further specified by electron microscopic in situ hybridization with a non-autoradiographic procedure.Our results indicate that the fibrillar centers are the sole nucleolar structures where rDNA, core histones, RNA polymerase I and DNA to po isomerase I are located together.Taking into account the potential value and limitations of immunoelectron microscopic techniques, we propose that transcription of the rRNA genes takes place within the confines of the fibrillar centers, probably close to the boundary regions to the surrounding dense fibrillar component.

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Section: Ill Immunolocaliza Non Of Histonesmentioning

confidence: 90%

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level

Thiry

Scheer

Goessens

1991

Electron Microscopy Reviews

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“…Immunofluorescence staining was carried out according to standard techniques. Antibodies used were anti-CF I m 68 rabbit polyclonal (1:500; Rü egsegger et al, 1998), anti-PSP1 rabbit peptide antibody (dilution 1:500), anti-PSF mouse monoclonal antibody (mAb) (dilution 1:500; Sigma-Aldrich), anti-fibrillarin mouse monoclonal 72b9 (dilution 1:50; Reimer et al, 1987), anti-SC35 mouse monoclonal (dilution 1:1000; Sigma-Aldrich), anti-SR proteins mouse monoclonal 16H3 (dilution 1:250; Zymed Laboratories, South San Francisco, CA), anti-p80 coilin monoclonal 5P10 (dilution 1:50), mouse monoclonal anti-CPSF100 (J1-27, Jenny et al, 1994), chicken anti-CstF64 (1:500), and fluorescein isothiocyanate (FITC)-and tetramethylrhodamine B isothiocyanate-conjugated secondary antibodies (FITC and Cy3, 1:500; Jackson ImmunoResearch, West Grove, PA). Before mounting on slides, coverslips were sometimes soaked in 1 g/ml 4Ј,6-diamidino-2-phenylindole (in 1ϫ PBS for 10 min) to stain DNA and/or followed by soaking in pyronin Y (0.66 mM in distilled H 2 O for 2 s) to stain RNA.…”

Section: Preparation Of Cell Lysates and Immunoblottingmentioning

confidence: 99%

Subnuclear Localization and Dynamics of the Pre-mRNA 3′ End Processing Factor Mammalian Cleavage Factor I 68-kDa Subunit

Cardinale

Cisterna

Bonetti

et al. 2007

MBoC

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Mammalian cleavage factor I (CF I m ) is an essential factor that is required for the first step in pre-mRNA 3 end processing. Here, we characterize CF I m 68 subnuclear distribution and mobility. Fluorescence microscopy reveals that in addition to paraspeckles CF I m 68 accumulates in structures that partially overlap with nuclear speckles. Analysis of synchronized cells shows that CF I m 68 distribution in speckles and paraspeckles varies during the cell cycle. At an ultrastructural level, CF I m 68 is associated with perichromatin fibrils, the sites of active transcription, and concentrates in interchromatin granules-associated zones. We show that CFI m 68 colocalizes with bromouridine, RNA polymerase II, and the splicing factor SC35. On inhibition of transcription, endogenous CF I m 68 no longer associates with perichromatin fibrils, but it can still be detected in interchromatin granules-associated zones. These observations support the idea that not only splicing but also 3 end processing occurs cotranscriptionally. Finally, fluorescence recovery after photobleaching analysis reveals that the CF I m 68 fraction associated with paraspeckles moves at a rate similar to the more dispersed molecules in the nucleoplasm, demonstrating the dynamic nature of this compartment. These findings suggest that paraspeckles are a functional compartment involved in RNA metabolism in the cell nucleus. INTRODUCTIONMost functional mRNAs of eukaryotic genes are generated from their primary transcripts (pre-mRNAs) through RNA splicing and 3Ј end polyadenylation. Removal of introns occurs in the spliceosome, a complex composed of five small ribonucleoprotein particles (U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles [snRNPs]) and many non-snRNPs splicing factors, including members of the arginine-serine (SR) family of proteins. The mature 3Ј ends of mRNAs are generated by endonucleolytic cleavage of the pre-mRNA followed by polyadenylation of the upstream cleavage product. Biochemical studies have identified six factors required for efficient processing in vitro: the cleavage and polyadenylation specificity factor (CPSF), the cleavage stimulation factor (CstF), and two cleavage factors, mammalian cleavage factor I m [CF I m ] and CF II m , are necessary for the cleavage reaction. Polyadenylation requires in addition to CPSF, poly(A) polymerase, and the nuclear poly(A) binding protein 1 (PABPN1, previously called PAB II, for review, see Wahle and Rü egsegger, 1999). Other proteins involved in either transcription, such as the carboxy-terminal domain of RNA polymerase II, or capping (nuclear cap-binding complex) and splicing (U2AF65) have been shown to greatly enhance the efficiency of the first step of the reaction (Flaherty et al., 1997;Hirose and Manley, 1998;Millevoi et al., 2002).The mammalian cell nucleus is a highly structured and dynamic compartment. Many nuclear factors are localized in morphologically well-defined structural units that include the nucleolus and several "nuclear bodies," such as the Cajal ...

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“…For example, small nuclear ribonucleoproteins (snRNPs) and the spliceosome were initially elucidated through the use of human autoantibodies (Lerner and Steitz, 1981;Tan, 1991). In addition, a number of unique nucleolar (Reimer et al, 1987), Golgi complex (Chan and Fritzler, 1998), and endosome (Waite et al, 1998;Selak et al, 1999) proteins have been identified using human autoantibodies.…”

Section: Introductionmentioning

confidence: 99%

A Phosphorylated Cytoplasmic Autoantigen, GW182, Associates with a Unique Population of Human mRNAs within Novel Cytoplasmic Speckles

Eystathioy

Chan

Tenenbaum

et al. 2002

MBoC

331

313

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A novel human cellular structure has been identified that contains a unique autoimmune antigen and multiple messenger RNAs. This complex was discovered using an autoimmune serum from a patient with motor and sensory neuropathy and contains a protein of 182 kDa. The gene and cDNA encoding the protein indicated an open reading frame with glycine-tryptophan (GW) repeats and a single RNA recognition motif. Both the patient's serum and a rabbit serum raised against the recombinant GW protein costained discrete cytoplasmic speckles designated as GW bodies (GWBs) that do not overlap with the Golgi complex, endosomes, lysosomes, or peroxisomes. The mRNAs associated with GW182 represent a clustered set of transcripts that are presumed to reside within the GW complexes. We propose that the GW ribonucleoprotein complex is involved in the posttranscriptional regulation of gene expression by sequestering a specific subset of gene transcripts involved in cell growth and homeostasis.

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Human autoantibodies: probes for nucleolus structure and function

Cited by 120 publications

References 72 publications

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level

Subnuclear Localization and Dynamics of the Pre-mRNA 3′ End Processing Factor Mammalian Cleavage Factor I 68-kDa Subunit

A Phosphorylated Cytoplasmic Autoantigen, GW182, Associates with a Unique Population of Human mRNAs within Novel Cytoplasmic Speckles

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