THE PATH OF TRANSCRIPTS FROM EXTRANUCLEOLAR SYNTHETIC SITES TO NUCLEAR-PORES - TRANSCRIPTS IN TRANSIT ARE CONCENTRATED IN DISCRETE STRUCTURES CONTAINING SR PROTEINS

The route taken by transcripts from synthetic sites in the nucleus to the cytoplasm has been under scrutiny for years, but details of the pa thway remain obscure. A new high-resolution method for mapping the pat hway is described; HeLa cells are grown in Br-U so that the analogue i s incorporated into RNA and exported to the cytoplasm, before Br-RNA i s localized by immune-electron microscopy, After exposure to low conce ntrations of Br-U for short periods, cells grow normally, Br-RNA is fi rst found in several thousand extra-nucleolar transcription sites or f actories (diameter 50-80 nm), before appearing in several hundred new downstream sites (diameter 50-80 nm) each minute; subsequently, progre ssively more downstream sites become labelled. These sites can be isol ated on sucrose gradients as large nuclear ribonucleoprotein particles of similar to 200 S. Later, Br-RNA is seen docked similar to 200 nm a way from similar to 20% nuclear pores, before exiting to the cytoplasm , Individual downstream sites are unlikely to contain individual trans cripts; rather, results are consistent with groups of transcripts bein g shipped together from synthetic sites to pores. A subset of SR prote ins are excellent markers of this pathway; this subset is concentrated in tens of thousands of sites, which include transcription, downstrea m and docking sites. Growth in high concentrations of Br-U for long pe riods is toxic, and Br-RNA accumulates just inside nuclear pores.