Microinjection of Anti-coilin Antibodies Affects the Structure of Coiled Bodies

doi:10.1083/jcb.142.4.899

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© The Rockefeller University Press, 0021-9525/1998//899 $5.00
The Journal of Cell Biology, Volume 142, Number 4, , 1998 899-912

Articles

Microinjection of Anti-coilin Antibodies Affects the Structure of Coiled Bodies

Fátima Almeida^*, Rainer Saffrich, Wilhelm Ansorge, and Maria Carmo-Fonseca^*

^* Institute of Histology and Embryology, Faculty of Medicine, University of Lisbon, 1699 Lisboa Codex, Portugal; and European Molecular Biology Laboratory, D-69117 Heidelberg, Germany

The coiled body is a distinct subnuclear domain enriched insmall nuclear ribonucleoprotein particles (snRNPs) involvedin processing of pre-mRNA. Although the function of the coiledbody is still unknown, current models propose that it may havea role in snRNP biogenesis, transport, or recycling. Here we describe that anti-coilin antibodies promote a specific disappearanceof the coiled body in living human cells, thus providing anovel tool for the functional analysis of this structure. Monoclonalantibodies (mAbs) were raised against recombinant human coilin,the major structural protein of the coiled body. Four mAbsare shown to induce a progressive disappearance of coiled bodies within $~$ 6 h after microinjection into the nucleus ofHeLa cells. After their disappearance, coiled bodies are notseen to re-form, although injected cells remain viable forat least 3 d. Epitope mapping reveals that the mAbs recognizedistinct amino acid motifs scattered along the complete coilinsequence. By 24 and 48 h after injection of antibodies thatpromote coiled body disappearance, splicing snRNPs are normallydistributed in the nucleoplasm, the nucleolus remains unaffected, and the cell cycle progresses normally. Furthermore, cellsdevoid of coiled bodies for $~$ 24 h maintain the ability to spliceboth adenoviral pre-mRNAs and transiently overexpressed humanβ-globin transcripts. In conclusion, within the time rangeof this study, no major nuclear abnormalities are detected aftercoiled body disappearance.

Key Words: coiled body • p80–coilin • splicing • spliceosomal snRNPs • nucleolus

Abbreviations used in this paper: Ad2, adenovirus type 2; BrdU, bromodeoxyuridine; BrUTP, 5-bromo-2'-uridine-5'-triphosphate; Cy5, indodicarbocyanine; MLP, major late promotor; NLS, nuclear localization signal; snoRNA, small nucleolar RNA; snRNP, small nuclear ribonucleoprotein.

Address all correspondence to M. Carmo-Fonseca, Institute ofHistology and Embryology, Faculty of Medicine, Av. Prof. EgasMoniz, 1699 Lisboa Codex, Portugal. Tel.: (351) 1 7934340.Fax: (351) 1 7951780. E-mail: hcarmo{at}fm.ul.pt

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