Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus.
Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus.
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DOI：
10.1083/jcb.122.2.283
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发表时间：
1993-07
期刊：
影响因子：
--
通讯作者：
de Jong L
中科院分区：
文献类型：
--
作者：
Wansink DG;Schul W;van der Kraan I;van Steensel B;van Driel R;de Jong L
若干核活动和组成部分集中在不连续的核隔室中。为了理解核区室化的功能意义，转录活性染色质的空间分布知识是必不可少的。我们研究了RNA聚合酶II（RPII）的转录位点的分布，通过5-溴尿苷5 '-三磷酸标记新生RNA，在体外和体内。新生RPII成绩单被发现在超过100个定义的区域，分散在整个核质。没有优惠本地化观察到无论是在核内部或周边。每个转录位点可以代表单个基因的活性，或者考虑到细胞中活性前mRNA基因的数量，代表一簇活性基因的活性。在双标记实验中，研究了新生RPII转录物的分布与必需剪接因子SC-35的分布之间的关系。抗SC-35的抗体除了标记这些结构域之间更弥散的区域之外，还识别许多明确的、强烈标记的核结构域（Spector，D. L.，X. -D. Fu和T.马尼亚蒂斯。1991. EMBO（Eur.摩尔Biol. Organ.）J. 10：3467-3481）。我们没有观察到强烈标记的SC-35域和前mRNA合成的网站之间的相关性。然而，RPII合成的许多位点与弱染色区域共定位。这意味着转录剪接发生在这些弱染色区域。这些区域也可能是剪接在转录后完成的位点。强烈标记的SC-35结构域可以作为剪接组分的组装、储存或再生的位点，或者作为内含子降解的隔室。
Several nuclear activities and components are concentrated in discrete nuclear compartments. To understand the functional significance of nuclear compartmentalization, knowledge on the spatial distribution of transcriptionally active chromatin is essential. We have examined the distribution of sites of transcription by RNA polymerase II (RPII) by labeling nascent RNA with 5-bromouridine 5'-triphosphate, in vitro and in vivo. Nascent RPII transcripts were found in over 100 defined areas, scattered throughout the nucleoplasm. No preferential localization was observed in either the nuclear interior or the periphery. Each transcription site may represent the activity of a single gene or, considering the number of active pre-mRNA genes in a cell, of a cluster of active genes. The relation between the distribution of nascent RPII transcripts and that of the essential splicing factor SC-35 was investigated in double labeling experiments. Antibodies against SC-35 recognize a number of well-defined, intensely labeled nuclear domains, in addition to labeling of more diffuse areas between these domains (Spector, D. L., X. -D. Fu, and T. Maniatis. 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:3467-3481). We observe no correlation between intensely labeled SC-35 domains and sites of pre-mRNA synthesis. However, many sites of RPII synthesis colocalize with weakly stained areas. This implies that contranscriptional splicing takes place in these weakly stained areas. These areas may also be sites where splicing is completed posttranscriptionally. Intensely labeled SC-35 domains may function as sites for assembly, storage, or regeneration of splicing components, or as compartments for degradation of introns.