Nucleoplasmic organization of small nuclear ribonucleoproteins in cultured human cells.
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.
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Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510.
Ward
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D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry*; D004247:DNA; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D015347:RNA Probes; D012326:RNA Splicing; D016372:RNA, Antisense; D012342:RNA, Small Nuclear / Q000032:analysis; D017411:Ribonucleoproteins, Small Nuclear / Q000032:analysis*; D017381:Spliceosomes / Q000737:chemistry; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D015347:RNA Probes; D016372:RNA, Antisense; D012342:RNA, Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D004247:DNA