Protein synthesis is required for rapid degradation of tubulin mRNA and other deflagellation-induced RNAs in Chlamydomonas reinhardi.

Abstract

After flagellar detachment in Chlamydomonas reinhardi, there is a rapid synthesis and accumulation of mRNAs for tubulin and other flagellar proteins. Maximum levels of these mRNAs (flagellar RNAs) are reached within 1 h after deflagellation, after which they are rapidly degraded to their predeflagellation levels. The degradation of alpha-and beta-tubulin RNAs was shown to be due to the shortening of their half-lives after accumulation (Baker et al., J. Cell Biol. 99:2074-2081. Deflagellation in the presence of protein synthesis inhibitors results in the accumulation of tubulin and other flagellar mRNAs by kinetics similar to those of controls. However, unlike controls, in which the accumulated mRNAs are rapidly degraded, these mRNAs are stabilized in cycloheximide. The stabilization by cycloheximide is specific for the flagellar mRNAs accumulated after deflagellation, since there is no change in the levels of flagellar mRNAs in nondeflagellated (uninduced) cells in the presence of cycloheximide. The kinetics of flagellar mRNA synthesis after deflagellation are shown to be the same in cycloheximide-treated and control cells by in vivo labeling and in vitro nuclear runoff experiments. These results show that protein synthesis is not required for the induced synthesis of flagellar mRNAs, and that all necessary transcriptional control factors are present in the cell before deflagellation, but that protein synthesis is required for the accelerated degradation of the accumulated flagellar mRNAs. Since cycloheximide prevents the induced synthesis and accumulation of flagellar proteins, it is possible that the product(s) of protein synthesis required for the accelerated decay of these mRNAs is a flagellar protein(s). The possibility that one or more flagellar proteins autoregulate the stability of the flagellar mRNAs is discussed.The cellular response to flagellar detachment in Chlamydomonas reinhardi includes a rapid and coordinate induction of the mRNAs for tubulin and other flagellar proteins (25,27,37,38). The accumulation of alpha-and beta-tubulin RNAs is accomplished by both a transient increase in transcription rate and an apparent stabilization of the tubulin transcripts during flagellar regeneration (3, 21). Maximum levels of mRNAs for tubulin and other flagellar proteins accumulate within 1 h, after which these RNAs are rapidly degraded (25,27,37,38). The half-life of tubulin mRNA during this deinduction is only about 20 min, or 2 to 3 times shorter than in nonregenerating, vegetative cells (3). These data suggest that a specific decay mechanism operates during deinduction which accelerates the return of the induced mRNAs to basal levels.In this report, we show that protein synthesis is not required for the activation of transcription and accumulation of tubulin and other induced mRNAs, but is necessary for the accelerated degradation of these RNAs after induction. MATERIALS AND METHODSCell growth and preparation. Chlamydomonas reinhardi vegetative cells, strain 21gr, were grown in medium I of Sager and Granic...

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“…The cloned products in the expected size range of 140 to 170 bp were used as probes for northern analyses, because genes encoding flagellar proteins can be identified after experimental deflagellation (Rosenbaum et al, 1969;Schloss et al, 1984) by a remarkably fast and strong transient accumulation of flagellar RNA caused by transcriptional induction as well as stabilization of flagellar RNA (Baker et al, 1986). One of our probes, pDIP, detected a small mRNA of 1.5 to 1.8 kb (Fig.…”

“…Plasmid DNA from E. coli, λDNA from lysates of recombinant phages, and DNA-fragments from agarose gels were prepared using commercially available purification systems (Qiagen, Hilden, Germany). RNA from C. reinhardtii was isolated according to Baker et al (Baker et al, 1986) and mRNA was isolated from total RNA using the oligotex dT mRNA purification system (Qiagen).…”

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.

“…Moreover, the decay rates of some individual mRNAs can vary as a consequence of differentiation (25,38,43), a particular stage of the cell cycle (79), or the presence of specific hormones (9) or specific inhibitors (3,22). The mechanisms responsible for determining either the intrinsic decay rate of an mRNA or the regulation of that rate are unknown.…”

Transcriptional regulation of coordinate changes in flagellar mRNAs during differentiation of Naegleria gruberi amebae into flagellates.

“…Tubulin RNA half-lives decrease two-to fourfold as flagella regenerate (1,2). The decline in tubulin mRNA stability is dependent on protein synthesis, although this requirement can apparently be circumvented by inducing reabsorption of the flagella, suggesting that a pool of tubulin or other flagellar proteins regulates flagellar tubulin mRNA stability (1).…”