In vivo equivalence of a cell-free system from rat liver for ribosomal RNA processing and transport.

The fidelity of release of ribosomal RNA from isolated nuclei has been studied in a reconstituted cell-free system derived from regenerating rat liver. The system consists of prelabeled nuclei incubated in homologous cytosol supplemented with salts, polyamines, dithiothreitol, buffers, methionine or S-adenosyhnethionine, ribonuclease inhibitor, ATP, and an energy-regenerating system. Processing of preformed precursor pools and release of mature 18 S and 28 S RNA occurs in the absence of transcription and shows an absolute requirement for labile cytosol proteins and energy as ATP. Release of this RNA parallels the decrease in the intranuclear precursor pools. In the cell-free system, the 45 S ribosomal precursor shows a half-life of 25 min at 30°C and 10 min at 37”C, which is identical with that observed in viva. The 32 to 28 S nuclear pool of ribosomal RNA is maintained until the 45 S precursor pool is depleted with essentially all of the potential 18 S and 28 S ribosomal RNA being conserved and released to the medium as mature ribosomal subunits. These subunits are active in in vitro protein synthesis. After a short in uiuo label, the relative specific activities of the in vitro transported 18 S and 28 S RNA reflect the relative sizes of the intranuclear precursor pools and support the intranuclear origin of the RNA. One of the early processing steps appears to be very temperaturesensitive and shows a sharp temperature transition about 25°C. It is concluded that the cell-free system reflects in uiuo events and is reliable for investigating ribosomal RNA processing and transport.