POST-TRANSCRIPTIONAL REGULATION OF R-PROTEIN SYNTHESIS

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These experiments are designed to characterize features of mRNAs which are determinants of translational regulation and mRNA stability. We will study the r-protein mRNAs of Dictyostelium which we have already shown to be selectively excluded from polysomes at the start of development and to undergo an apparent decrease in stability later in development. 1) Translational regulation: The relative translational efficiency of r-protein mRNAs will be assessed in in vitro systems programmed with increasing amounts of total poly(A)+ RNA, with either initiation or elongation made rate limiting by the addition of inhibitors. Experiments to identify sequences which are responsible for translational discrimination in vivo will utilize recombinant plasmids in which regions of genes which encode mRNAs with different translational efficiencies in development have been exchanged. Translation of the chimeric mRNAs expressed from these plasmids will be examined in transformed, developing cells. In addition, expression of r-protein mRNAs from a promoter active in development will allow us to ask whether post-transcriptional modifications which may change with mRNA "age" are important in determining translatability in developing cells. The cellular location of untranslated, but stable, r-protein mRNAS will be examined by in situ hybridization to developing cells. 2) mRNA stability: The time and magnitude of the developmental change in r-protein mRNA stability will be determined by measurement of mRNA transcription (in nuclear run-off assays) and mRNA half-life (in pulse-chase experiments). Events known to affect mRNA stability in Dictyostelium will be correlated with the decrease in relative abundance of r-protein mRNAs. The importance of specific sequences and of efficient translation during development will be examined using chimeric transcripts from plasmids introduced by transformation. We will examine by in situ hybridization whether changes in stability are correlated with changes in subcellular location. Finally, we will try to identify a nuclease activity responsible for stage specific degradation of r-protein mRNAS in developmental cell lysates.