Preservation of specific RNA distribution within the chromatin-depleted nuclear substructure demonstrated by in situ hybridization coupled with biochemical fractionation
UMass Chan Affiliations
Department of Cell BiologyDocument Type
Journal ArticlePublication Date
1991-03-01Keywords
Cell Line; Cell Nucleolus; Cell Nucleus; Cell Transformation, Viral; DNA Replication; Herpesvirus 4, Human; Humans; Nucleic Acid Hybridization; RNA; RNA, Viral; Thymidine; Transcription, GeneticCell Biology
Life Sciences
Medicine and Health Sciences
Metadata
Show full item recordAbstract
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.Source
J Cell Biol. 1991 Mar;112(6):1055-63. Link to article on publisher's websiteDOI
10.1083/jcb.112.6.1055Permanent Link to this Item
http://hdl.handle.net/20.500.14038/34082PubMed ID
1705562Related Resources
Link to Article in PubMedae974a485f413a2113503eed53cd6c53
10.1083/jcb.112.6.1055