Microinjection of anti-coilin antibodies affects the structure of coiled bodies.
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.
Institute of Histology and Embryology, Faculty of Medicine, University of Lisbon, 1699 Lisboa Codex, Portugal.
F
F
Almeida
R
R
Saffrich
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W
Ansorge
M
M
Carmo-Fonseca
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002453:Cell Cycle / Q000502:physiology; D002466:Cell Nucleolus / Q000378:metabolism; D018604:Epitope Mapping; D005455:Fluorescent Antibody Technique; D005914:Globins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008845:Microinjections; D009687:Nuclear Proteins / Q000276:immunology / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics; D011994:Recombinant Proteins / Q000276:immunology; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
D000911:Antibodies, Monoclonal; D009687:Nuclear Proteins; D012322:RNA Precursors; D011994:Recombinant Proteins; D017411:Ribonucleoproteins, Small Nuclear; C068921:p80-coilin; D005914:Globins