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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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Coiled body disappearance does not impair  splicing. (A–D) HeLa cells  were microinjected with  mAb-δ, incubated for either 6  (A and B) or 24 h (C and D)  and then infected with Ad2  for 18 h. The cells were fixed  and hybridized with an oligonucleotide probe complementary to the first splice  junction of the tripartite  leader (A and C). The injected antibody was detected  using a secondary antibody  conjugated to Texas red (B  and D), and the viral protein  DBP was detected using a  secondary antibody conjugated to Cy5 (data not  shown). The hybridization  pattern in the injected cells  (A, arrow; and C) is similar to  that of noninjected cells (A,  arrowheads). Note that in B  and D, staining of the nucleoplasm is nonhomogeneous.  This is due to the very intense  signal produced by anti-DBP  antibody, which is partially  detected in the Texas red  channel of the confocal microscope. (E and F) Cells  were microinjected with mAb  1D4-δ, incubated for 24 h,  and then re-injected with a  expression plasmid carrying  the human β-globin gene.  The cells were fixed 16 h later  and hybridized with an oligonucleotide probe complementary to the first splice junction of β-globin mRNA (E). The injected antibody  was detected using a secondary IgG conjugated to Texas red (F). Note that under the mild permeabilization conditions used here, the  hybridization signal is predominantly detected in the cytoplasm. Bar, 10 μm.
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Figure 10: Coiled body disappearance does not impair splicing. (A–D) HeLa cells were microinjected with mAb-δ, incubated for either 6 (A and B) or 24 h (C and D) and then infected with Ad2 for 18 h. The cells were fixed and hybridized with an oligonucleotide probe complementary to the first splice junction of the tripartite leader (A and C). The injected antibody was detected using a secondary antibody conjugated to Texas red (B and D), and the viral protein DBP was detected using a secondary antibody conjugated to Cy5 (data not shown). The hybridization pattern in the injected cells (A, arrow; and C) is similar to that of noninjected cells (A, arrowheads). Note that in B and D, staining of the nucleoplasm is nonhomogeneous. This is due to the very intense signal produced by anti-DBP antibody, which is partially detected in the Texas red channel of the confocal microscope. (E and F) Cells were microinjected with mAb 1D4-δ, incubated for 24 h, and then re-injected with a expression plasmid carrying the human β-globin gene. The cells were fixed 16 h later and hybridized with an oligonucleotide probe complementary to the first splice junction of β-globin mRNA (E). The injected antibody was detected using a secondary IgG conjugated to Texas red (F). Note that under the mild permeabilization conditions used here, the hybridization signal is predominantly detected in the cytoplasm. Bar, 10 μm.

Mentions: In this study, cells were microinjected with mAb 1D4-δ, incubated for either 6 or 24 h, and then infected with Ad2 for 18 h (Fig. 10, A–D). The cells were fixed and hybridized with an oligonucleotide probe complementary to the first splice junction of the tripartite leader (Bridge et al., 1996). To unambiguously identify cells that were simultaneously injected and infected, a triple-labeling experiment was performed using rhodamine to detect the injected mAb, Cy5 for the viral protein DBP (Linné et al., 1977), and fluorescein for spliced viral mRNA. As shown in Fig. 10, A and B, the hybridization signal produced by the splice junction probe is similar in injected (Fig. 10 A, arrow) and noninjected cells (Fig. 10 A, arrowheads). Similar results were observed on cells microinjected with mAb 1D4-δ, incubated for 24 h, and then infected with Ad2 for 18 h (Fig. 10, C and D). Similar results were also observed after injection of mAbs 3W8-o (which also promotes disappearance of coiled bodies) and 5P10-π (which does not disassemble coiled bodies; data not shown). Immunofluorescence analysis of injected cells using antibodies against the penton protein further confirmed that late viral gene expression was active in these cells (data not shown).


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

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

Coiled body disappearance does not impair  splicing. (A–D) HeLa cells  were microinjected with  mAb-δ, incubated for either 6  (A and B) or 24 h (C and D)  and then infected with Ad2  for 18 h. The cells were fixed  and hybridized with an oligonucleotide probe complementary to the first splice  junction of the tripartite  leader (A and C). The injected antibody was detected  using a secondary antibody  conjugated to Texas red (B  and D), and the viral protein  DBP was detected using a  secondary antibody conjugated to Cy5 (data not  shown). The hybridization  pattern in the injected cells  (A, arrow; and C) is similar to  that of noninjected cells (A,  arrowheads). Note that in B  and D, staining of the nucleoplasm is nonhomogeneous.  This is due to the very intense  signal produced by anti-DBP  antibody, which is partially  detected in the Texas red  channel of the confocal microscope. (E and F) Cells  were microinjected with mAb  1D4-δ, incubated for 24 h,  and then re-injected with a  expression plasmid carrying  the human β-globin gene.  The cells were fixed 16 h later  and hybridized with an oligonucleotide probe complementary to the first splice junction of β-globin mRNA (E). The injected antibody  was detected using a secondary IgG conjugated to Texas red (F). Note that under the mild permeabilization conditions used here, the  hybridization signal is predominantly detected in the cytoplasm. Bar, 10 μm.
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Figure 10: Coiled body disappearance does not impair splicing. (A–D) HeLa cells were microinjected with mAb-δ, incubated for either 6 (A and B) or 24 h (C and D) and then infected with Ad2 for 18 h. The cells were fixed and hybridized with an oligonucleotide probe complementary to the first splice junction of the tripartite leader (A and C). The injected antibody was detected using a secondary antibody conjugated to Texas red (B and D), and the viral protein DBP was detected using a secondary antibody conjugated to Cy5 (data not shown). The hybridization pattern in the injected cells (A, arrow; and C) is similar to that of noninjected cells (A, arrowheads). Note that in B and D, staining of the nucleoplasm is nonhomogeneous. This is due to the very intense signal produced by anti-DBP antibody, which is partially detected in the Texas red channel of the confocal microscope. (E and F) Cells were microinjected with mAb 1D4-δ, incubated for 24 h, and then re-injected with a expression plasmid carrying the human β-globin gene. The cells were fixed 16 h later and hybridized with an oligonucleotide probe complementary to the first splice junction of β-globin mRNA (E). The injected antibody was detected using a secondary IgG conjugated to Texas red (F). Note that under the mild permeabilization conditions used here, the hybridization signal is predominantly detected in the cytoplasm. Bar, 10 μm.
Mentions: In this study, cells were microinjected with mAb 1D4-δ, incubated for either 6 or 24 h, and then infected with Ad2 for 18 h (Fig. 10, A–D). The cells were fixed and hybridized with an oligonucleotide probe complementary to the first splice junction of the tripartite leader (Bridge et al., 1996). To unambiguously identify cells that were simultaneously injected and infected, a triple-labeling experiment was performed using rhodamine to detect the injected mAb, Cy5 for the viral protein DBP (Linné et al., 1977), and fluorescein for spliced viral mRNA. As shown in Fig. 10, A and B, the hybridization signal produced by the splice junction probe is similar in injected (Fig. 10 A, arrow) and noninjected cells (Fig. 10 A, arrowheads). Similar results were observed on cells microinjected with mAb 1D4-δ, incubated for 24 h, and then infected with Ad2 for 18 h (Fig. 10, C and D). Similar results were also observed after injection of mAbs 3W8-o (which also promotes disappearance of coiled bodies) and 5P10-π (which does not disassemble coiled bodies; data not shown). Immunofluorescence analysis of injected cells using antibodies against the penton protein further confirmed that late viral gene expression was active in these cells (data not shown).

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