Limits...
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.

Show MeSH

Related in: MedlinePlus

Coiled body disappearance does  not affect snRNP localization. HeLa cells  were microinjected with mAb-δ and incubated for either 24 (A–D) or 48 h (E and  F) before fixation. The injected antibody  was detected using a secondary antibody  conjugated to Texas red (A, C, and E).  The cells were double-labeled with either  an antisense riboprobe specific for U2 snRNA (B), an antibody against the Sm proteins (D), or an antisense riboprobe specific for U3 snoRNA (F). Arrows in B  point to U2 snRNA concentrated in the  coiled bodies of noninjected cells. Bar,  10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132868&req=5

Figure 9: Coiled body disappearance does not affect snRNP localization. HeLa cells were microinjected with mAb-δ and incubated for either 24 (A–D) or 48 h (E and F) before fixation. The injected antibody was detected using a secondary antibody conjugated to Texas red (A, C, and E). The cells were double-labeled with either an antisense riboprobe specific for U2 snRNA (B), an antibody against the Sm proteins (D), or an antisense riboprobe specific for U3 snoRNA (F). Arrows in B point to U2 snRNA concentrated in the coiled bodies of noninjected cells. Bar, 10 μm.

Mentions: Having established that intranuclear injection of mAbs promotes the disappearance of coiled bodies, we next asked how depriving a cell of the coiled body affects the subcellular distribution of splicing snRNPs. The U1, U2, U5, and U4/U6 snRNPs, all of which are localized in the coiled body, represent major components of the spliceosome. Each of these snRNPs is built of a unique species of snRNA associated with specific proteins. Additionally, all splicing snRNPs share a common set of proteins, termed the Sm proteins, that are tightly bound to the snRNAs (for review see Moore et al., 1993). During their biogenesis, the U1, U2, U4, and U5 snRNAs are transcribed in the nucleus and transported to the cytoplasm, where the Sm proteins bind. Thereafter, the assembled snRNP is imported to the nucleus (for review see Mattaj, 1988). In the nucleus, snRNPs are normally distributed throughout the nucleoplasm with additional concentration in interchromatin granules and coiled bodies (for review see Lamond and Carmo-Fonseca, 1993). To investigate whether the coiled body plays an important role in the subcellular distribution of snRNPs, cells were microinjected with mAb 1D4-δ, incubated for 24 h, fixed, and then double-labeled with either an antisense riboprobe specific for U2 snRNA (Fig. 9, A and B) or an antibody against the Sm proteins (Fig. 9, C and D). As depicted in the figure, no apparent change is observed in the distribution of either U2 snRNA or Sm protein in injected cells. Namely, there is no evidence for an accumulation of snRNA or Sm protein in the cytoplasm, and within the nucleus the distribution appears normal, except for the lack of accumulation at coiled bodies which is typically observed in noninjected cells (Fig. 9 B, arrows). Similar results were observed on cells microinjected with mAb 1D4-δ and incubated for 48 h before fixation and labeling (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 affect snRNP localization. HeLa cells  were microinjected with mAb-δ and incubated for either 24 (A–D) or 48 h (E and  F) before fixation. The injected antibody  was detected using a secondary antibody  conjugated to Texas red (A, C, and E).  The cells were double-labeled with either  an antisense riboprobe specific for U2 snRNA (B), an antibody against the Sm proteins (D), or an antisense riboprobe specific for U3 snoRNA (F). Arrows in B  point to U2 snRNA concentrated in the  coiled bodies of noninjected cells. Bar,  10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132868&req=5

Figure 9: Coiled body disappearance does not affect snRNP localization. HeLa cells were microinjected with mAb-δ and incubated for either 24 (A–D) or 48 h (E and F) before fixation. The injected antibody was detected using a secondary antibody conjugated to Texas red (A, C, and E). The cells were double-labeled with either an antisense riboprobe specific for U2 snRNA (B), an antibody against the Sm proteins (D), or an antisense riboprobe specific for U3 snoRNA (F). Arrows in B point to U2 snRNA concentrated in the coiled bodies of noninjected cells. Bar, 10 μm.
Mentions: Having established that intranuclear injection of mAbs promotes the disappearance of coiled bodies, we next asked how depriving a cell of the coiled body affects the subcellular distribution of splicing snRNPs. The U1, U2, U5, and U4/U6 snRNPs, all of which are localized in the coiled body, represent major components of the spliceosome. Each of these snRNPs is built of a unique species of snRNA associated with specific proteins. Additionally, all splicing snRNPs share a common set of proteins, termed the Sm proteins, that are tightly bound to the snRNAs (for review see Moore et al., 1993). During their biogenesis, the U1, U2, U4, and U5 snRNAs are transcribed in the nucleus and transported to the cytoplasm, where the Sm proteins bind. Thereafter, the assembled snRNP is imported to the nucleus (for review see Mattaj, 1988). In the nucleus, snRNPs are normally distributed throughout the nucleoplasm with additional concentration in interchromatin granules and coiled bodies (for review see Lamond and Carmo-Fonseca, 1993). To investigate whether the coiled body plays an important role in the subcellular distribution of snRNPs, cells were microinjected with mAb 1D4-δ, incubated for 24 h, fixed, and then double-labeled with either an antisense riboprobe specific for U2 snRNA (Fig. 9, A and B) or an antibody against the Sm proteins (Fig. 9, C and D). As depicted in the figure, no apparent change is observed in the distribution of either U2 snRNA or Sm protein in injected cells. Namely, there is no evidence for an accumulation of snRNA or Sm protein in the cytoplasm, and within the nucleus the distribution appears normal, except for the lack of accumulation at coiled bodies which is typically observed in noninjected cells (Fig. 9 B, arrows). Similar results were observed on cells microinjected with mAb 1D4-δ and incubated for 48 h before fixation and labeling (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