NUCLEOPLASMIC ORGANIZATION OF SMALL NUCLEAR RIBONUCLEOPROTEINS IN CULTURED HUMAN-CELLS
NUCLEOPLASMIC ORGANIZATION OF SMALL NUCLEAR RIBONUCLEOPROTEINS IN CULTURED HUMAN-CELLS
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DOI：
10.1083/jcb.121.4.715
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发表时间：
1993-05-01
影响因子：
7.8
通讯作者：
WARD, DC
中科院分区：
文献类型：
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作者：
MATERA, AG;WARD, DC
用荧光原位杂交技术，用反义DNA和2‘-O-Me RNA寡核苷酸，研究了8种小核糖核蛋白(U1、U2、U4、U5和U6 RNAs以及另外3种SnRNAs U11、U12和7SK)在体外培养的人细胞中的组成。利用高灵敏的数字成像显微镜，我们证明了所有这些SnRNA都广泛分布在核质中，但它们被排除在核仁之外。此外，U2、U4、U5、U6和U12单链RNA集中在离散的核团中，称为盘状小体，而U1和7SK不集中。除了盘状小体外，在单层培养的HeLa细胞的核浆中还观察到了经典的斑点图案，而悬浮生长的HeLa细胞则显示出更弥漫的核质标记。用不同的SnRNP特异性抗血清进行的免疫荧光染色显示，与它们的反义SnRNA寡核苷酸对应物完全一致。虽然U2 RNA集中在盘状小体中，但对U2反义探针的荧光信号的定量检测表明，U2 SnRNP的大部分位于核质中。此外，同时显示U2SnRNAs和简单重复的U2基因表明盘状小体不是U2转录的位置。
The organization of eight small nuclear ribonucleoproteins (the U1, U2, U4, U5, and U6 RNAs previously studied by others and three additional snRNAs, U11, U12, and 7SK) has been investigated in cultured human cells by fluorescence in situ hybridization with antisense DNA and 2'-O-Me RNA oligonucleotides. Using highly sensitive digital imaging microscopy we demonstrate that all of these snRNAs are widespread throughout the nucleoplasm, but they are excluded from the nucleoli. In addition, the U2, U4, U5, U6, and U12 snRNAs are concentrated in discrete nuclear foci, known as coiled bodies, but U1 and 7SK are not. In addition to coiled bodies, a classic speckled pattern was observed in the nucleoplasm of monolayer-grown HeLa cells, whereas suspension-grown HeLa cells revealed a more diffuse nucleoplasmic labeling. Immunofluorescence staining using various snRNP-specific antisera shows complete agreement with that of their antisense snRNA oligonucleotide counterparts. Although U2 RNA is concentrated in coiled bodies, quantitation of the fluorescence signals from the U2 antisense probe reveals that the bulk of the U2 snRNP is located in the nucleoplasm. Furthermore, simultaneous visualization of the U2 snRNAs and the tandemly repeated U2 genes demonstrates that coiled bodies are not the sites of U2 transcription.