Partial purification of a developmentally regulated messenger RNA from Dictyostelium discoideum by thermal elution from poly(U)-Sepharose

Abstract

We have demonstrated that thermal elution from poly(U)-Sepharose is useful for separating newly synthesized messenger RNAs from steady-state mRNAs isolated from vegetative cells of Dictyostelium discoideum (Palatnik et al., 1979,1980). Because we were able to enrich between ten- and 60-fold for new transcripts, we suggested that this technique might be applicable to the purification of developmentally regulated mRNAs. The utility of the technique would, however, depend on the differential rate of synthesis of the particular mRNAs. For example, in vegetatively growing Dictyostelium amoebae the relative distribution of translation activities in newly synthesized mRNA is similar to that found in steady-state mRNA (Palatnik et al., 1979), and the technique does not lead to substantial purification of any particular mRNA species. In order to test the applicability of the method, we have studied, as a model system, changes in actin mRNA synthesis during early D. discoideum development. It has been shown that the relative concentration of actin mRNA changes during Dictyostelium development. Experiments with inhibitors of RNA synthesis have suggested that changes in the concentration of this mRNA are mediated at the transcriptional level. This conclusion is supported by the experiments of this paper: newly synthesized poly(A)-containing RNA from growing and developing cells was translated in mRNA-dependent reticulocyte lysates and the translation products were analyzed by two-dimensional polyacrylamide gel electrophoresis. At two hours of development, when the relative concentration of actin mRNA is at its peak, we find that its translation activity is also substantially enriched in the fraction of RNA that is newly synthesized. At five hours of development, when the percentage of total translatable actin mRNA has begun to decline, actin represents a much smaller percentage of the translation products coded for by this RNA fraction. The physical isolation and characterization of the newly synthesized mRNA fraction thus provides evidence that changes in the concentration of actin mRNA during Dictyostelium development occur at the level of messenger RNA synthesis. Furthermore, the results dramatically illustrate the usefulness of thermal elution from poly(U)-Sepharose for enriching for mRNAs induced by alterations in developmental or metabolic states.