Polyamine Regulation of Ornithine Decarboxylase Synthesis in Neurospora crassa

Abstract

Ornithine decarboxylase (ODC) of the fungus Neurospora crassa, encoded by the spe-1 gene, catalyzes an initial and rate-limiting step in polyamine biosynthesis and is highly regulated by polyamines. In N. crassa, polyamines repress the synthesis and increase the degradation of ODC protein. Changes in the rate of ODC synthesis correlate with similar changes in the abundance of spe-1 mRNA. We identify two sequence elements, one in each of the 5 and 3 regions of the spe-1 gene of N. crassa, required for this polyamine-mediated regulation. A 5 polyamine-responsive region (5 PRR) comprises DNA sequences both in the upstream untranscribed region and in the long 5 untranslated region (5-UTR) of the gene. The 5 PRR is sufficient to confer polyamine regulation to a downstream, heterologous coding region. Use of the ␤-tubulin promoter to drive the expression of various portions of the spe-1 transcribed region revealed a 3 polyamine-responsive region (3 PRR) downstream of the coding region. Neither changes in cellular polyamine status nor deletion of sequences in the 5-UTR alters the half-life of spe-1 mRNA. Sequences in the spe-1 5-UTR also impede the translation of a heterologous coding region, and polyamine starvation partially relieves this impediment. The results show that N. crassa uses a unique combination of polyamine-mediated transcriptional and translational control mechanisms to regulate ODC synthesis.Ornithine decarboxylase (ODC) catalyzes an initial, ratelimiting reaction in the biosynthesis of the polyamines, the conversion of the amino acid ornithine to the divalent polyamine putrescine (1,4-diaminobutane). Putrescine undergoes two subsequent aminopropyl transfer reactions, in which it is converted first to the trivalent spermidine and then to the tetravalent spermine. Polyamines are essential for the growth of normal, and particularly neoplastic, cell types (25, 38), but excess spermidine and spermine are toxic (8,28).ODC is a one of the most highly regulated enzymes of eukaryotic cells, its activity varying over a 100-fold range. ODC activity responds to extracellular signals such as mitogens and growth factors and to changes in the intracellular concentrations of the polyamines themselves. Polyamine regulation of ODC activity is unusual in that the end products do not act as allosteric effectors of this initial enzyme but control only the synthesis and degradation of the ODC protein (reviewed in references 6 and 41).In the filamentous fungus Neurospora crassa, ODC is encoded by the spe-1 gene (3, 9). The regulation of ODC in this fungus resembles that in other eukaryotic organisms. Polyamines reduce the rate of synthesis and increase the rate of degradation of ODC protein (1). However, unlike all other eukaryotes in which polyamine-mediated regulation has been studied, changes in the rate of synthesis ODC in N. crassa are correlated with similar changes (ca. 10-fold) in the abundance of spe-1 mRNA (43). Previously, we identified two regions of the spe-1 gene that affect its expression (27). An upstream ac...

Spermidine Is a Morphogenetic Determinant for Cell Fate Specification in the Male Gametophyte of the Water FernMarsilea vestita

“…The translation of these newly unmasked transcripts explains the rapid and dramatic rise in detectable levels of spermidine that precedes later maturation phases of the spermatids (Figure 1). In other organisms, polyamines have been implicated in the regulation of their own biosynthesis pathways (Hoyt et al, 2000). The large rise in spermidine concentration late in development can be blocked by the addition of CHA at a point when the spermatids are already present, thereby showing that the enzyme is responsible for the synthesis of the polyamine.…”

uORFs with unusual translational start codons autoregulate expression of eukaryotic ornithine decarboxylase homologs

“…Studies on the 5Ј leader of ODC mRNA in Neurospora crassa (36), showed that a region that we now recognize contains the uCC in Pezizomycotina (Fig. 1Bvi), causes close to 30-fold repression of expression of ODC protein.…”

Control of mRNA Stability in Fungi by NMD, EJC and CBC Factors Through 3′UTR Introns

“…Only wt cells labeled with 4TU gave a strong signal over background ( Figure S3, A and B). As expected, new transcription was necessary to obtain 4TU-labeled RNA: preincubation with the general transcription inhibitor thiolutin, which has been shown to decrease transcription in N. crassa (Hoyt et al 2000;Lee et al 2009), severely reduced 4TU incorporation, as did chilling cells on ice ( Figure S3, C and D). Incorporation of 4TU into RNA rapidly diminished when excess uracil was added to cultures ( Figure S3E) and 4TU itself had minimal impact on N. crassa growth under the experimental conditions used (,30 min incubation) or during growth for extended periods ( Figure S3F), similar to its minimal effects on growth in S. cerevisiae (Munchel et al 2011) and S. pombe (Sun et al 2012).…”