The path of transcripts from extra-nucleolar synthetic sites to nuclear pores: transcripts in transit are concentrated in discrete structures containing SR proteins

Abstract

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.