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. 1999 May;10(5):1653-63.
doi: 10.1091/mbc.10.5.1653.

Coiled bodies preferentially associate with U4, U11, and U12 small nuclear RNA genes in interphase HeLa cells but not with U6 and U7 genes

E Y Jacobs  1 , M R Frey, W Wu, T C Ingledue, T C Gebuhr, L Gao, W F Marzluff, A G Matera
Affiliations
Free PMC article

Coiled bodies preferentially associate with U4, U11, and U12 small nuclear RNA genes in interphase HeLa cells but not with U6 and U7 genes

E Y Jacobs et al. Mol Biol Cell. 1999 May.
Free PMC article

Abstract

Coiled bodies (CBs) are nuclear organelles involved in the metabolism of small nuclear RNAs (snRNAs) and histone messages. Their structural morphology and molecular composition have been conserved from plants to animals. CBs preferentially and specifically associate with genes that encode U1, U2, and U3 snRNAs as well as the cell cycle-regulated histone loci. A common link among these previously identified CB-associated genes is that they are either clustered or tandemly repeated in the human genome. In an effort to identify additional loci that associate with CBs, we have isolated and mapped the chromosomal locations of genomic clones corresponding to bona fide U4, U6, U7, U11, and U12 snRNA loci. Unlike the clustered U1 and U2 genes, each of these loci encode a single gene, with the exception of the U4 clone, which contains two genes. We next examined the association of these snRNA genes with CBs and found that they colocalized less frequently than their multicopy counterparts. To differentiate a lower level of preferential association from random colocalization, we developed a theoretical model of random colocalization, which yielded expected values for chi2 tests against the experimental data. Certain single-copy snRNA genes (U4, U11, and U12) but not controls were found to significantly (p < 0.000001) associate with CBs. Recent evidence indicates that the interactions between CBs and genes are mediated by nascent transcripts. Taken together, these new results suggest that CB association may be substantially augmented by the increased transcriptional capacity of clustered genes. Possible functional roles for the observed interactions of CBs with snRNA genes are discussed.

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Figures

Figure 1
Figure 1
Chromosomal locations of snRNA and histone genes used in this study. Map locations of the single-copy snRNA genes (determined by RH and cytogenetic mapping) as well as those of previously characterized snRNA and histone loci are shown. Bars to the right of the ideograms show the range of FISH signals. The nearest markers from the RH mapping are shown to the right of the chromsomes; MIT framework markers are shown in bold.
Figure 2
Figure 2
Sequence comparison of human and mouse RNU7 loci. The U7 genes at human 12p13 (accession number U474924) and mouse chromosome 6F (X54165) along with flanking regions are compared. Note the relatively high degree of upstream similarity, including the functionally conserved control regions called the proximal and distal sequence elements (PSE and DSE, overlined). The human U7 snRNA coding region is also overlined. A single-nucleotide difference between the published RNA sequence (accession number M17910) and the human 12p13 sequence is illustrated. Downstream regions, with the exception of a putative 3′ box terminator sequence (overlined), are not conserved.
Figure 3
Figure 3
The human U7 sequence at 12p13 encodes a functional snRNA. (A) Xenopus oocytes were injected with H20 (lanes 2 and 3), anti-hnRNP A1 mRNA control oligonucleotide (lanes 4 and 5), or anti-U7 snRNA oligonucleotide (lanes 6 and 7). Four hours later, oocytes were injected with either a control plasmid and the mouse H2a-614 gene (lanes 2, 4, and 6) or plasmid pTT005, containing the putative human U7 gene (hU7) and the mouse H2a-614 gene (lanes 3, 5, and 7). Eighteen hours later, total RNA was prepared from oocytes and analyzed by S1 nuclease mapping using an assay that detects both processed (Proc) and unprocessed (Unproc) histone mRNA. NS, nonspecific transcript derived from the hU7 plasmid. Lane 1, pUC18 DNA digested with HpaII. (B) Schematic of the S1 nuclease assay used in A. The probe contains 34 nucleotides beyond the end of the histone mRNA followed by plasmid sequences that are not present in the transcript from the mouse histone H2a-614 gene.
Figure 4
Figure 4
Single-copy snRNA loci also colocalize with CBs in interphase HeLa cells. Nuclei were stained with DAPI (blue); CBs are displayed in red; and snRNA gene signals are in green. In A (RNU11) and B (RNU12), BAC clones corresponding to the appropriate locus were used to generate the FISH signals; CB immunofluorescence was performed, and cells were scored as described previously (Frey and Matera, 1995). In each panel, a typical colocalization event is shown; the full data set is presented in Table 1.
Figure 5
Figure 5
Diagram showing a nucleus with CBs, locus signals, a nucleolus, and a CB–locus association. The dashed line around the CB represents the effective area of a CB that is available for colocalization if locus signals are considered points. The nucleoplasmic area is essentially equal to the area of the nucleus minus the nucleolar area. Average values were nuclear area, 133.7 μm2; nucleolar area, 10.9 μm2; nucleoplasmic area (AN), 123 μm2; and effective CB area (Acb), 0.66 μm2.
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