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Microinjection of anti-coilin antibodies affects the structure of coiled bodies.

Almeida F, Saffrich R, Ansorge W, Carmo-Fonseca M - J. Cell Biol. (1998)

Bottom Line: After their disappearance, coiled bodies are not seen to re-form, although injected cells remain viable for at least 3 d.Epitope mapping reveals that the mAbs recognize distinct amino acid motifs scattered along the complete coilin sequence.Furthermore, cells devoid of coiled bodies for approximately 24 h maintain the ability to splice both adenoviral pre-mRNAs and transiently overexpressed human beta-globin transcripts.

View Article: PubMed Central - PubMed

Affiliation: Institute of Histology and Embryology, Faculty of Medicine, University of Lisbon, 1699 Lisboa Codex, Portugal.

ABSTRACT
The coiled body is a distinct subnuclear domain enriched in small nuclear ribonucleoprotein particles (snRNPs) involved in processing of pre-mRNA. Although the function of the coiled body is still unknown, current models propose that it may have a role in snRNP biogenesis, transport, or recycling. Here we describe that anti-coilin antibodies promote a specific disappearance of the coiled body in living human cells, thus providing a novel tool for the functional analysis of this structure. Monoclonal antibodies (mAbs) were raised against recombinant human coilin, the major structural protein of the coiled body. Four mAbs are shown to induce a progressive disappearance of coiled bodies within approximately 6 h after microinjection into the nucleus of HeLa cells. After their disappearance, coiled bodies are not seen to re-form, although injected cells remain viable for at least 3 d. Epitope mapping reveals that the mAbs recognize distinct amino acid motifs scattered along the complete coilin sequence. By 24 and 48 h after injection of antibodies that promote coiled body disappearance, splicing snRNPs are normally distributed in the nucleoplasm, the nucleolus remains unaffected, and the cell cycle progresses normally. Furthermore, cells devoid of coiled bodies for approximately 24 h maintain the ability to splice both adenoviral pre-mRNAs and transiently overexpressed human beta-globin transcripts. In conclusion, within the time range of this study, no major nuclear abnormalities are detected after coiled body disappearance.

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Epitope mapping of mAbs. (A) Diagram showing the reactivity of each mAb with the  full-length coilin and different deletion mutants  of the protein, as determined by immunoblot  analysis. The data show that the epitopes recognized by mAbs o, φ, and γ map between amino  acids 1–127, 187–291, and 292–362, respectively.  The mAbs δ and pδ react with epitopes located  between acids 363 and 481. The mAb-π recognizes either an epitope localized around amino  acids 481–482, or a conformational epitope  present in the coilin mutant encompassing amino  acids 363–576, but absent from the mutants encompassing amino acids 363–481 and 482–576.  (B) Diagram showing the mapped epitopes (𝕐) in  relation to the complete coilin sequence. Hatched  regions depict the two motifs that closely match  the consensus sequence of simple and bipartite  nuclear localization sequences, NLSa and NLSb,  respectively (Bohmann et al., 1995a). The diagram also depicts the position of serine 202. Conversion of this amino acid residue into aspartate  causes the disappearance of coiled bodies and a  redistribution of coilin to intranucleolar domains  (Lyon et al., 1997).
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Figure 2: Epitope mapping of mAbs. (A) Diagram showing the reactivity of each mAb with the full-length coilin and different deletion mutants of the protein, as determined by immunoblot analysis. The data show that the epitopes recognized by mAbs o, φ, and γ map between amino acids 1–127, 187–291, and 292–362, respectively. The mAbs δ and pδ react with epitopes located between acids 363 and 481. The mAb-π recognizes either an epitope localized around amino acids 481–482, or a conformational epitope present in the coilin mutant encompassing amino acids 363–576, but absent from the mutants encompassing amino acids 363–481 and 482–576. (B) Diagram showing the mapped epitopes (𝕐) in relation to the complete coilin sequence. Hatched regions depict the two motifs that closely match the consensus sequence of simple and bipartite nuclear localization sequences, NLSa and NLSb, respectively (Bohmann et al., 1995a). The diagram also depicts the position of serine 202. Conversion of this amino acid residue into aspartate causes the disappearance of coiled bodies and a redistribution of coilin to intranucleolar domains (Lyon et al., 1997).

Mentions: To obtain monoclonal antibodies, hybridomas were derived by fusion of the mouse myeloma cell line Ag8.653 with spleen cells from Balb/c mice immunized with recombinant human coilin. Six clones (designated δ, pδ, γ, π, o, and φ) were isolated. The antibodies secreted by clones δ, pδ, o, and φ are of the IgG1 class, whereas mAb-γ is IgG2b and mAb-π is IgG2a (Table I). Immunoblot analysis reveals that all mAbs recognize a band of ∼80 kD in HeLa protein extracts (Fig. 1, A and B). Clones π and δ react specifically with p80–coilin, whereas the other clones cross-react with additional peptides. In addition to coilin, the mAbs o and γ recognize a major cytoplasmic protein band of ∼140 kD, mAb-φ strongly reacts with a nuclear protein of ∼110 kD, and mAb-pδ reveals a minor nuclear protein band of ∼115 kD. To map the epitopes recognized by each mAb, a series of His–coilin deletion mutants was generated and probed by immunoblot analysis (Fig. 2). From these data we conclude that the different clones react with epitopes distributed along the entire protein sequence. By immunofluorescence all mAbs labeled coiled bodies (Fig. 3 A and data not shown), as previously reported for clone δ (Rebelo et al., 1996).


Microinjection of anti-coilin antibodies affects the structure of coiled bodies.

Almeida F, Saffrich R, Ansorge W, Carmo-Fonseca M - J. Cell Biol. (1998)

Epitope mapping of mAbs. (A) Diagram showing the reactivity of each mAb with the  full-length coilin and different deletion mutants  of the protein, as determined by immunoblot  analysis. The data show that the epitopes recognized by mAbs o, φ, and γ map between amino  acids 1–127, 187–291, and 292–362, respectively.  The mAbs δ and pδ react with epitopes located  between acids 363 and 481. The mAb-π recognizes either an epitope localized around amino  acids 481–482, or a conformational epitope  present in the coilin mutant encompassing amino  acids 363–576, but absent from the mutants encompassing amino acids 363–481 and 482–576.  (B) Diagram showing the mapped epitopes (𝕐) in  relation to the complete coilin sequence. Hatched  regions depict the two motifs that closely match  the consensus sequence of simple and bipartite  nuclear localization sequences, NLSa and NLSb,  respectively (Bohmann et al., 1995a). The diagram also depicts the position of serine 202. Conversion of this amino acid residue into aspartate  causes the disappearance of coiled bodies and a  redistribution of coilin to intranucleolar domains  (Lyon et al., 1997).
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Related In: Results  -  Collection

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Figure 2: Epitope mapping of mAbs. (A) Diagram showing the reactivity of each mAb with the full-length coilin and different deletion mutants of the protein, as determined by immunoblot analysis. The data show that the epitopes recognized by mAbs o, φ, and γ map between amino acids 1–127, 187–291, and 292–362, respectively. The mAbs δ and pδ react with epitopes located between acids 363 and 481. The mAb-π recognizes either an epitope localized around amino acids 481–482, or a conformational epitope present in the coilin mutant encompassing amino acids 363–576, but absent from the mutants encompassing amino acids 363–481 and 482–576. (B) Diagram showing the mapped epitopes (𝕐) in relation to the complete coilin sequence. Hatched regions depict the two motifs that closely match the consensus sequence of simple and bipartite nuclear localization sequences, NLSa and NLSb, respectively (Bohmann et al., 1995a). The diagram also depicts the position of serine 202. Conversion of this amino acid residue into aspartate causes the disappearance of coiled bodies and a redistribution of coilin to intranucleolar domains (Lyon et al., 1997).
Mentions: To obtain monoclonal antibodies, hybridomas were derived by fusion of the mouse myeloma cell line Ag8.653 with spleen cells from Balb/c mice immunized with recombinant human coilin. Six clones (designated δ, pδ, γ, π, o, and φ) were isolated. The antibodies secreted by clones δ, pδ, o, and φ are of the IgG1 class, whereas mAb-γ is IgG2b and mAb-π is IgG2a (Table I). Immunoblot analysis reveals that all mAbs recognize a band of ∼80 kD in HeLa protein extracts (Fig. 1, A and B). Clones π and δ react specifically with p80–coilin, whereas the other clones cross-react with additional peptides. In addition to coilin, the mAbs o and γ recognize a major cytoplasmic protein band of ∼140 kD, mAb-φ strongly reacts with a nuclear protein of ∼110 kD, and mAb-pδ reveals a minor nuclear protein band of ∼115 kD. To map the epitopes recognized by each mAb, a series of His–coilin deletion mutants was generated and probed by immunoblot analysis (Fig. 2). From these data we conclude that the different clones react with epitopes distributed along the entire protein sequence. By immunofluorescence all mAbs labeled coiled bodies (Fig. 3 A and data not shown), as previously reported for clone δ (Rebelo et al., 1996).

Bottom Line: After their disappearance, coiled bodies are not seen to re-form, although injected cells remain viable for at least 3 d.Epitope mapping reveals that the mAbs recognize distinct amino acid motifs scattered along the complete coilin sequence.Furthermore, cells devoid of coiled bodies for approximately 24 h maintain the ability to splice both adenoviral pre-mRNAs and transiently overexpressed human beta-globin transcripts.

View Article: PubMed Central - PubMed

Affiliation: Institute of Histology and Embryology, Faculty of Medicine, University of Lisbon, 1699 Lisboa Codex, Portugal.

ABSTRACT
The coiled body is a distinct subnuclear domain enriched in small nuclear ribonucleoprotein particles (snRNPs) involved in processing of pre-mRNA. Although the function of the coiled body is still unknown, current models propose that it may have a role in snRNP biogenesis, transport, or recycling. Here we describe that anti-coilin antibodies promote a specific disappearance of the coiled body in living human cells, thus providing a novel tool for the functional analysis of this structure. Monoclonal antibodies (mAbs) were raised against recombinant human coilin, the major structural protein of the coiled body. Four mAbs are shown to induce a progressive disappearance of coiled bodies within approximately 6 h after microinjection into the nucleus of HeLa cells. After their disappearance, coiled bodies are not seen to re-form, although injected cells remain viable for at least 3 d. Epitope mapping reveals that the mAbs recognize distinct amino acid motifs scattered along the complete coilin sequence. By 24 and 48 h after injection of antibodies that promote coiled body disappearance, splicing snRNPs are normally distributed in the nucleoplasm, the nucleolus remains unaffected, and the cell cycle progresses normally. Furthermore, cells devoid of coiled bodies for approximately 24 h maintain the ability to splice both adenoviral pre-mRNAs and transiently overexpressed human beta-globin transcripts. In conclusion, within the time range of this study, no major nuclear abnormalities are detected after coiled body disappearance.

Show MeSH
Related in: MedlinePlus