The production of ribosomal RNA from high molecular weight precursors I. Factors which influence the ability of isolated nucleoli to process 45-S RNA

Abstract

A search was made for conditions which would allow isolated nucleoli to convert 45-S RNA in vitro to 28-S and 18-S ribosomal RNA or to other natural derivatives. The nuclease activity of nucleoli purified from L cells may be altered or controlled as follows: (a) if incubation of nucleoli in vitro is preceded by a wash with relatively high concentrations (about 100 μg/ml) of polyvinyl sulfate or with 0.5 M NaCl and deoxyribonuclease, nuclease activity is almost totally suppressed; (b) for extensive degradation of 45-S RNA to occur Mg 2+ must be present in the incubation medium; and (c) inclusion of bulk quantities of RNA in the incubation medium allows the selective breakdown of 45-S RNA without the production of heterogeneous fragments. After incubation for 30 min at 30° in a medium containing 0.5 mM Mg 2+ and 100–200 μg RNA per ml, 45-S nucleolar RNA was degraded, and a limited quantity of fragments which migrated in acrylamide gels like the 32-S component and another intermediate were produced. Under these conditions extrinsic 45-S RNA which was incubated together with the nucleoli was also degraded, but extrinsic 32-S, 28-S and 18-S RNA underwent little or no degradation. The fragments produced during incubation in vitro were enriched in their methyl group/uridine ratio as compared to the 45-S component, although the enrichment was not as great as that observed after conversion in vivo. These results suggest a tentative model for the conversion process whereby 45-S RNA undergoes a single scission with an endonuclease followed by a stepwise trimming with exonuclease.