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The route taken by transcripts from synthetic sites in the nucleus to the cytoplasm has been under scrutiny for years, but details of the pathway remain obscure. A new high-resolution method for mapping the pathway is described; HeLa cells are grown in Br-U so that the analogue is incorporated into RNA and exported to the cytoplasm, before Br-RNA is localized by immuno-electron microscopy. After exposure to low concentrations of Br-U for short periods, cells grow normally. Br-RNA is first found in several thousand extra-nucleolar transcription sites or factories (diameter 50-80 nm), before appearing in several hundred new downstream sites (diameter 50-80 nm) each minute; subsequently, progressively more downstream sites become labelled. These sites can be isolated on sucrose gradients as large nuclear ribonucleoprotein particles of approximately 200 S. Later, Br-RNA is seen docked approximately 200 nm away from approximately 20% nuclear pores, before exiting to the cytoplasm. Individual downstream sites are unlikely to contain individual transcripts; rather, results are consistent with groups of transcripts being shipped together from synthetic sites to pores. A subset of SR proteins are excellent markers of this pathway; this subset is concentrated in tens of thousands of sites, which include transcription, downstream and docking sites. Growth in high concentrations of Br-U for long periods is toxic, and Br-RNA accumulates just inside nuclear pores.

Type

Journal article

Journal

J Cell Sci

Publication Date

08/1998

Volume

111 ( Pt 15)

Pages

2269 - 2282

Keywords

Cell Nucleolus, Cell Nucleus, Cytoplasm, HeLa Cells, Humans, Immunohistochemistry, Microscopy, Immunoelectron, Nuclear Envelope, RNA, Messenger, RNA-Binding Proteins, Sodium Chloride, Transcription, Genetic, Uridine