Preservation of specific RNA distribution within the chromatin-depleted nuclear substructure demonstrated by in situ hybridization coupled with biochemical fractionation

doi:10.1083/jcb.112.6.1055

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Preservation of specific RNA distribution within the chromatin-depleted nuclear substructure demonstrated by in situ hybridization coupled with biochemical fractionation

YG Xing and JB Lawrence
Department of Cell Biology, University of Massachusetts Medical School, Worcester 01655.

Biochemical fractionation procedures previously shown to remove 95% of cellular protein, DNA, and phospholipid, were combined with fluorescence in situ hybridization to provide a critical evaluation of the retention and spatial preservation of specific primary transcripts within the chromatin-depleted nuclear substructure, operationally defined as the nuclear "matrix." This unique approach made it possible to directly address whether nuclear extraction procedures preserve, create, or destroy ribonucleoprotein filament structures. Comparison of nuclei before and after fractionation demonstrated that localized foci or "tracks" of specific nRNA are unambiguously retained in the nuclear matrix preparation. Two well-characterized nuclear fractionation procedures were used and three Epstein-Barr virus-infected cell types investigated, including latently and permissively infected cells carrying integrated or episomal genomes. The EBV primary transcripts as well as nucleolar RNA were preserved within the remaining nuclear substructure with unambiguous spatial and quantitative fidelity. Image processing and quantitative microfluorimetry, together with [3H]thymidine labeling of DNA, show that essentially 100% of the RNA signal intensity remained after removal of 85% of the DNA. That the native RNA distribution was unchanged was shown in other experiments in which the same individual nRNA tracks were examined before and after fractionation. Results conclusively demonstrate that the tight restriction of RNA to highly localized sites is independent of bulk DNA removal and of extensive extraction of proteins and phospholipids. Hence, this work provides direct visual evidence that the primary transcripts studied are localized via their binding to, or comprising part of, non-chromatin nuclear substructure.
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Janevski, J., Park, P. C., De Boni, U. (1997). Changes in Morphology and Spatial Position of Coiled Bodies during NGF-induced Neuronal Differentiation of PC12 Cells. J. Histochem. Cytochem. 45: 1523-1532 [Abstract] [Full Text]
Nickerson, J. A., Krockmalnic, G., Wan, K. M., Penman, S. (1997). The nuclear matrix revealed by eluting chromatin from a cross-linked�nucleus. Proc. Natl. Acad. Sci. USA 94: 4446-4450 [Abstract] [Full Text]
Panning, B, Jaenisch, R (1996). DNA hypomethylation can activate Xist expression and silence X-linked genes.. Genes Dev. 10: 1991-2002 [Abstract]
Dirks, R., Daniel, K., Raap, A. (1995). RNAs radiate from gene to cytoplasm as revealed by fluorescence in situ hybridization. J. Cell Sci. 108: 2565-2572 [Abstract]
Meller, V., Fisher, P. (1995). Nuclear distribution of Drosophila DNA topoisomerase II is sensitive to both RNase and DNase. J. Cell Sci. 108: 1651-1657 [Abstract]
Xing, Y, Johnson, C., Dobner, P., Lawrence, J. (1993). Higher level organization of individual gene transcription and RNA splicing. Science 259: 1326-1330 [Abstract]
Carter, K., Bowman, D, Carrington, W, Fogarty, K, McNeil, J., Fay, F., Lawrence, J. (1993). A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus. Science 259: 1330-1335 [Abstract]
Sahlas, D., Milankov, K, Park, P., De Boni, U (1993). Distribution of snRNPs, splicing factor SC-35 and actin in interphase nuclei: immunocytochemical evidence for differential distribution during changes in functional states. J. Cell Sci. 105: 347-357 [Abstract]
Cremer, T., Kurz, A., Zirbel, R., Dietzel, S., Rinke, B., Schrock, E., Speicher, M.R., Mathieu, U., Jauch, A., Emmerich, P., Scherthan, H., Ried, T., Cremer, C., Lichter, P. (1993). Role of Chromosome Territories in the Functional Compartmentalization of the Cell Nucleus. Cold Spring Harb Symp Quant Biol 58: 777-792 [Abstract]
Davis, I., Francis-Lang, H., Ish-Horowicz, D. (1993). Mechanisms of Intracellular Transcript Localization and Export in Early Drosophila Embryos. Cold Spring Harb Symp Quant Biol 58: 793-798 [Abstract]
Lawrence, J.B., Carter, K.C., Xing, X. (1993). Probing Functional Organization within the Nucleus: Is Genome Structure Integrated with RNA Metabolism?. Cold Spring Harb Symp Quant Biol 58: 807-818 [Abstract]
Nuovo, G J, Gorgone, G A, MacConnell, P, Margiotta, M, Gorevic, P D (1992). In situ localization of PCR-amplified human and viral cDNAs.. Genome Res 2: 117-123 [Abstract]
Amberg, D C, Goldstein, A L, Cole, C N (1992). Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA.. Genes Dev. 6: 1173-1189 [Abstract]
Huang, S, Spector, D L (1991). Nascent pre-mRNA transcripts are associated with nuclear regions enriched in splicing factors.. Genes Dev. 5: 2288-2302 [Abstract]