Localization and kinetics of formation of nuclear heterodisperse RNA, cytoplasmic heterodisperse RNA and polyribosome-associated messenger RNA in HeLa cells

Abstract

Three heterogeneous RNA species in HeLa cells have been characterized with respect to kinetics of labeling, turnover and location. These are the nucleoplasmic, cytoplasmic and polyribosome-associated messenger fractions. The heterogeneous nucleoplasmic RNA is found entirely in the nucleoplasm if sufficently high ionic strength buffer and zonal sucrose density centrifugation are used in the nuclear fractionation. The RNA is isolated in the form of particulates with sedimentation coefficients up to 5000 s. Nucleoplasmic RNA synthesis is shown to be relatively unaffected by doses of actinomycin which completely inhibit ribosomal RNA synthesis. The labeling of the nucleoplasmic RNA indicates that it turns over rapidly with a mean life of approximately one hour. The rate of decay of the nucleoplasmic RNA in the presence of a high concentration of actinomycin is initially similar to the rate of formation of this RNA, but subsequently becomes aberrant. A heterogeneous RNA is found associated with the cytoplasm using the fractionation techniques described. Its kinetics of labeling suggest that it is unrelated to the nucleoplasmic RNA. Only a portion of the total cytoplasmic RNA is associated with polyribosomes. Polyribosomes are unaffected by the level of actinomycin used to inhibit ribosomal RNA synthesis for at least seven hours. Labeled messenger RNA can be isolated from polyribosomes free of contamination by newly synthesized ribosomal RNA. A heterogeneous RNA which co-sediments with, but is not attached to, polyribosomes is identified. Polyribosome-attached messenger RNA has a range of sedimentation values from 8 to 30 s with a maximum at about 18 s, whereas the contaminating RNA is broadly distributed from 10 to 70 s. Agents which alter the sedimentation of polyribosomes in preparative sucrose gradients, such as puromycin and EDTA, also alter the distribution of messenger RNA in a similar manner but leave the non-messenger RNA unaffected. The kinetics of appearance of messenger RNA indicate that it is probably not derived from the nucleoplasmic heterogeneous RNA. The existence of a pool is inferred which results in a 15-minute lag before labeled messenger RNA appears on polyribosomes, and which continues to supply messenger for 20 minutes after the administration of a high concentration of actinomycin.