Role of protein synthesis in decay and accumulation of mRNA during spore germination in the cellular slime mold Dictyostelium discoideum.

Abstract

Spore germination in Dictyostelium discoideum is a particularly suitable model for studying the regulation of gene expression, since developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. The previous isolation of three cDNA clones specific for mRNA developmentally regulated during spore germination allowed for the quantitation of the specific itlRNAs during this process. The three mRNAs specific to clones pLK109, pLK229, and pRK270 have half-lives much shorter (minutes) than those of constitutive mRNAs (hours). Using spore germination as a model, we studied the roles of ribosome-mRNA interactions and protein synthesis in mRNA degradation by using antibiotics that inhibit specific reactions in protein biosynthesis. Cycloheximide inhibits the elongation step of protein synthesis. Polysomes accumulate in inhibited cells because ribosomes do not terminate normally and new ribosomes enter the polysome, eventually saturating the mRNA. Pactamycin inhibits initiation, and consequently polysomes break down in the presence of this drug. Under this condition, the mRNA is essentially free of ribosomes. pLK109, pLK229, and pRK270 mRNAs were stabilized in the presence of cycloheximide, but pactamycin had no effect on their normal decay. Since it seems likely that stability of mRNA reflects the availability of sites for inactivation by nucleases, it follows that in the presence of cycloheximide, these sites are protected, presumably by occupancy by ribosomes. No ribosomes are bound to mRNA in the presence of pactamycin, and therefore mRNA degrades at about the normal rate. The data further indicate that a labile protein is probably not involved in mRNA decay or stabilization, since protein synthesis is inhibited equally by both antibiotics. We conclude that it may be important to use more than one type of protein synthesis inhibitor to evaluate whether protein synthesis is required for mRNA decay. The effect of protein synthesis ihhibition on mRNA synthesis and accumulation was also studied. mRNA synthesis continues in the presence of the inhibitors, albeit at a diminished rate relative to that of the uninhibited control.

Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

“…A role for translational efficiency in the regulation of mRNA stability is suggested by the destabilizing effects of drugs which disrupt mRNAribosome association (1,33), premature translational termination codons (41,45), and clustered rare codons (26). For all three types of effects it has been postulated that, when ribosome association is diminished or eliminated, mRNA degradation is enhanced by the increased access of specific nucleases to the mRNA.…”

“…It is likely that these complex decay kinetics reflect the sum of the decay rates of individual mRNAs (e.g., in D. discoideum, the most stable mRNAs have half-lives of approximately 10 h and the least stable mRNAs have half-lives which are approximately 10-fold shorter). It has been suggested that such large differences in the stability of individual mRNAs could be accounted for by differences in their respective sizes (39,52,58,63,78,81), poly(A) tail lengths (8,28,56,91), ribosome loading (1,26,33,41,45), or 5'-and 3'-untranslated (UT) sequences (42,53,64,69,74,76,89). To test the validity of these hypotheses, we have identified cloned cDNAs which encode mRNAs that are representative of the stability extremes in D. discoideum and have initiated a characterization of the properties of the respective mRNAs.…”

Effect of protein synthesis inhibition on gene expression during early development of Dictyostelium discoideum.

“…Development was initiated by removing bacteria or nutrient broth, followed by plating the cells as described previously (31). Cycloheximide, when used, was present at 400 ,ug/ml, and pactamycin (gift of The Upjohn Co., Kalamazoo, Mich.) was used at 300 jig/ml (17). Inhibition of protein synthesis was determined as outlined by Finney et al (11).…”

The stability of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells varies with growth rate.

“…The stabilization of these short-lived mRNAs by either inhibitors of initiation or elongation indicates a requirement for continuing protein synthesis in the decay of these mRNAs. However, the mRNAs for tyrosine aminotransferase (42), vesicular stomatitis (33), and several proteins of Dictyostelium discoideum (22) were found to be stable in the presence of cycloheximide but not in the presence of pactamycin or puromycin. These later findings have suggested that shielding of mRNA by polysomes (which occurs when elongation but not initiation is inhibited) may protect against mRNA decay (3,22,37).…”

“…However, the mRNAs for tyrosine aminotransferase (42), vesicular stomatitis (33), and several proteins of Dictyostelium discoideum (22) were found to be stable in the presence of cycloheximide but not in the presence of pactamycin or puromycin. These later findings have suggested that shielding of mRNA by polysomes (which occurs when elongation but not initiation is inhibited) may protect against mRNA decay (3,22,37). Although the use of translational inhibitors has failed to provide evidence for a general mechanism for mRNA degradation, it has accentuated the importance of interactions of mRNA with components of the translational apparatus in the destabilization of mRNA.…”