Independent mechanisms are utilized for the coordinate and transient accumulation of two differentiation-specific mrnas during differentiation of naegleria gruberi amoebae into flagellates

During the differentiation of Naegleria gruberi amoebae into flagellates, four differentiation-specific (DS) mRNAs are transiently and coordinately accumulated. Three of the four DS mRNAs, Class II, III, and IV, encode α-tubulin, β-tubulin, and flagellar calmodulin, respectively. The protein product of the Class I mRNA has not been identified. We examined the effects of inhibition of protein synthesis on transcription and accumulation of β-tubulin mRNA and Class I mRNA to understand the mechanism of coordinate regulation. Inhibition of protein synthesis at the beginning of differentiation completely blocked transcription of the β-tubulin gene. Addition of cycloheximide at 30 or 40 min after initiation of differentiation inactivated transcription of the β-tubulin gene in less than 10 min as judged by nuclear run-on experiments. However, once differentiation had proceeded for more than 50 min, inhibition of protein synthesis did not inactivate transcription of the β-tubulin gene. Rather, transcription of β-tubulin mRNA was more active in cycloheximide-treated cells than in control cells. Cycloheximide treatment at the initiation of the differentiation also blocked transcription of the Class I gene. However, addition of the drug after 30 min had no significant effect on the transcription of the Class I gene. Cycloheximide treatment also increased the half-lives of β-tubulin and Class I mRNA drastically. These data suggest that: (1) the transient accumulation of the two DS mRNAs during differentiation are regulated by changing both the rate of transcription and the stability of the mRNAs; (2) protein synthesis is required for the transcriptional and post-transcriptional regulations; (3) the transcriptional regulation mechanism of the β-tubulin gene and that of the Class I gene are distinct; and (4) the transcription of the β-tubulin gene is regulated by different mechanisms during differentiation.