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

Abstract

The nuclear run-on technique was used to measure the rate of transcription of flagellar genes during the differentiation of Naegleria gruberi amebae into flagellates. Synthesis of mRNAs for the axonemal proteins aand l-tubulin and flagellar calmodulin, as wel as a coordinately regulated poly(A)+ RNA that codes for an unidentified protein, showed transient increases averaging 22-fold. The rate of synthesis of two poly(A)+ RNAs common to amebae and flagellates was low until the transcription of the flagellar genes began to decline, at which time synthesis of the RNAs found in amebae increased 3-to 10-fold. The observed changes in the rate of transcription can account quantitatively for the 20-fold increase in flagellar mRNA concentration during the differentiation. The data for the flageilar calmodulin gene demonstrate transcriptional regulation for a nontubulin axonemal protein. The data also demonstrate at least two programs of transcriptional regulation during the differentiation and raise the intriguing possibility that some significant fraction of the nearly 200 different proteins of the flagellar axoneme is transcriptionally regulated during the 1 h it takes N. grubeni amebae to form visible flagella.Axonemes of eucaryotic flagella are composed of nearly 200 different proteins (28). Thus, neglecting proteins associated with basal bodies and the various rootlet structures found in most cells, the formation of cilia or flagella requires the synthesis and assembly of a minimum of 200 gene products. This complexity is particularly notable because some cells can assemble flagella quite rapidly.The best-studied example of flagellum formation is the biflagellated green alga Chlamydomonas reinhardtii. Most studies of flagellum formation in C. reinhardtii have taken advantage of the fact that the cells are readily deflagellated to examine the regeneration of flagella (e.g., see references 27, 32, 33, and 39). Regeneration involves both the assembly of preexisting flagellar proteins, which can produce about 50% of the original flagellar length, and the synthesis of new protein (33). Transient increases in a number of mRNAs coding for flagellar proteins have been documented during C. reinhardi flagellar regeneration (34), but changes in only two of these RNAs have been studied at the transcriptional level. Changes in the level of mRNAs for the a and ,B subunits of tubulin were found to be due, in part, to changes in the rate of their synthesis and, in part, to changes in tubulin mRNA stability (2,19). No data are available to suggest how proteins that are exclusively flagellar are regulated (C. reinhardtii contains only one form of ,-tubulin which presumably must be used in cytoplasmic and mitotic microtubules as well as flagella) (40). It is also of interest to ask whether the regulatory mechanisms governing regeneration of flagella in a normally flagellated cell would apply to a cell forming flagella de novo.The amebo-flagellate Naegleria gruberi provides an opportunity to examine this question. In contrast to C. rei...

Effects of retinoic acid and cAMP on the differentiation ofNaegleria gruberiamoebas into flagellates

“…A cDNA fragment of each mRNA (Pst I fragment of pcNg 8-5, 580 bp, for 3-tubulin; PCR fragment of 380 bp, for actin; Pst I fragment of pcNg 3-28, 700 bps, for a non-specific mRNA [Lee and Walsh, 1988]) was labeled with 35 P-dCTP by using a DNA labeling kit (Boehringer Mannheim).…”

“…At 30 min after the initiation, the rate of transcription of DS genes is about 90% of the peak rate (Lee and Walsh, 1988) and the concentration of DS mRNAs is 40% of the maximum and rapidly increasing (Mar et al, 1986). Addition of RA at 30 min significantly inhibited differentiation.…”

“…O-O, control; A-A, RA at Omin. peak rate (50-70%, Lee and Walsh, 1988), the concentration of DS mRNAs is at the maximum (Mar et al, 1986), and protein synthesis required for the formation of the flagellar apparatus has been completed (Walsh, 1984). Addition of RA at 70 min did not inhibit the formation of flagella, but the formation of the flagellated shape was still inhibited.…”

“…At the end of differentiation (120 min), the concentrations of these mRNAs are below 20% of their respective maximum values (Mar et al, 1986). This transient accumulation of the three mRNAs is due, in part, to transient transcriptional activation of the respective genes after the initiation of differentiation (Lee and Walsh, 1988). Transcription of these three genes is not detected in amoebas, but activated rapidly after the initiation of differentiation.…”

“…At 70 min when the concentration of the mRNAs is at the maximum, the rate of transcription of these genes has already decreased to 50-70% of the peak rate. At the end of differentiation (120 min), these genes are transcribed slowly, below 10-20% of the peak rate (Lee and Walsh, 1988).…”