Microinjection of anti-coilin antibodies affects the structure of coiled bodies
Microinjection of anti-coilin antibodies affects the structure of coiled bodies
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DOI：
10.1083/jcb.142.4.899
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发表时间：
1998-08-24
影响因子：
7.8
通讯作者：
Carmo-Fonseca, M
中科院分区：
文献类型：
--
作者：
Almeida, F;Saffrich, R;Carmo-Fonseca, M
卷曲体是一个独特的亚核结构域，富含参与前体mRNA加工的小核核糖核蛋白颗粒（snRNP）。虽然卷曲体的功能仍然是未知的，目前的模型提出，它可能在snRNP生物合成，运输或回收中发挥作用。在这里，我们描述了抗卷曲蛋白抗体促进活的人类细胞中卷曲体的特异性消失，从而为这种结构的功能分析提供了一种新的工具。针对重组人卷曲蛋白（卷曲体的主要结构蛋白）产生单克隆抗体（mAb）。研究表明，四种mAb在体内诱导卷曲体逐渐消失，类似于显微注射到HeLa细胞核后6小时。在它们消失后，没有看到卷曲体重新形成，尽管注射的细胞保持活力至少3天。表位作图显示，mAb识别沿着完整卷曲蛋白序列分散的不同氨基酸基序。注射促进卷曲体消失的抗体后24和48小时，剪接snRNP正常分布在核质中，核仁保持不受影响，细胞周期正常进行。此外，缺乏卷曲体的细胞保持类似于24小时的剪接腺病毒前体mRNA和瞬时过表达的人β-珠蛋白转录物的能力。总之，在本研究的时间范围内，在卷曲体消失后未检测到重大核异常。
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,similar to 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 similar to 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.