Polyamine regulation of ribosome pausing at the upstream open reading frame of S-adenosylmethionine decarboxylase.

Synthesis of S-adenosylmethionine decarboxylase (AdoMetDC), a key regulated enzyme in the pathway of polyamine biosynthesis, is feedback-controlled at the level of translation by spermidine and spermine. The peptide product of an upstream open reading frame (uORF) in the mRNA is solely responsible for polyamine regulation of AdoMetDC translation. Using a primer extension inhibition assay and in vitro protein synthesis reactions, we found ribosomes paused at or close to the termination codon of the uORF. This pause was greatly diminished with the altered uORFs' sequences that abolish uORF regulation in vivo. The half-life of the ribosome pause was related to the concentration of polyamines present but was unaffected by magnesium concentration. Furthermore, inhibition of translation initiation at a reporter gene placed downstream of the AdoMetDC uORF directly correlated with the stability of the ribosome pause at the uORF. These observations are consistent with a model in which regulation of ribosome pausing at the uORF by polyamines controls ribosome access to the downstream AdoMetDC reading frame.

In vitro translation of the upstream open reading frame in the mammalian mRNA encoding S-adenosylmethionine decarboxylase.

The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive element that suppresses translation of the associated downstream cistron in vivo. In this paper, we provide the first direct evidence of peptide synthesis from the S-adenosylmethionine decarboxylase uORF using an in vitro translation system. We examine both the influence of cation concentration on peptide synthesis and the effect of altering the uORF sequence on peptide synthesis. Synthesis of wild type and altered peptides was similar at all concentrations of magnesium tested. In contrast, synthesis of the wild type peptide was more sensitive than that of altered peptides to elevated concentrations of the naturally occurring polyamines, spermidine and spermine, as well as several polyamine analogs. The sensitivity of in vitro synthesis to spermidine was influenced by both the amino acid sequence and the length of the peptide product of the uORF. Findings from the present study correlate with the effects of the uORF and polyamines on translation of a downstream cistron in vivo and support the hypothesis that polyamines and the structure of the nascent peptide create a rate-limiting step in uORF translation, perhaps through a ribosome stalling mechanism.

ZBP-99 defines a conserved family of transcription factors and regulates ornithine decarboxylase gene expression.

Among transcription factors that regulate ornithine decarboxylase (ODC) gene expression are those that interact with GC-rich promoters, including Sp1 and ZBP-89. Sp1 functions as a transactivator and ZBP-89 as a transrepressor of both the ODC and gastrin promoters. This study reports the cloning and characterization of a second member of the ZBP family that also binds GC boxes. ZBP-99 contains four Krüppel-type zinc fingers that collectively share 91% amino acid sequence similarity and 79% sequence identity with those found in ZBP-89. In addition, there are highly conserved amino acid sequences in the carboxy-terminal segments of the two genes. In spite of their structural similarities, the two proteins are encoded at distinct loci, ZBP-89 on chromosome 3q21 and ZBP-99 on 1q32.1. The predicted open reading frame of ZBP-99 cDNA encodes a 99-kDa protein. Electrophoretic mobility shift assays showed that ZBP-99 protein specifically binds to the GC-rich promoter elements of gastrin and ODC genes. Northern blot analysis showed that a major ZBP-99 transcript of 5.6 kb is expressed ubiquitously at low levels, with elevated expression levels in placenta and in adult kidney, liver, and lymphocytes. Cotransfection of AGS gastric adenocarcinoma and HT-29 colon adenocarcinoma cells with a ZBP-99 expression construct and with an ODC reporter construct show that ZBP-99 repressed basal expression in the two cell lines by 80 and 60%, respectively. Collectively, the data suggest that ZBP-99 binds GC-rich promoters and may complement the activities mediated by ZBP-89.

Ornithine decarboxylase is a transcriptional target of tumor suppressor WT1.

The product of the Wilm's tumor suppressor gene, WT1, is a zinc-finger DNA-binding protein, which is thought to be a transcription factor. Two genes, those encoding epidermal growth factor receptor and syndecan-1, are known to be endogenous targets of WT1. Previous studies had identified binding sites for WT1 in the promoter of the ornithine decarboxylase (ODC) gene. In this paper, we tested whether the endogenous ODC gene might be a target of WT1 by establishing lines of baby hamster kidney (BHK) cells that expressed WT1 isoform A under control of a tetracycline-regulated expression system. When expression of WT1 was activated in BHK cells, the cellular level of ODC mRNA declined, with kinetics that correlated with the increase in WT1 level, demonstrating that the endogenous ODC gene was indeed responsive to cellular level of WT1. WT1 isoforms A and B inhibited the activity of the ODC promoter by approximately fivefold in transiently transfected BHK cells, while isoforms C and D, which have altered DNA binding domains, had no significant effect. The sequence CTCCCCCGC, located at nucleotides -106 to -98 relative to the site of transcriptional initiation in the ODC gene, interacted with the zinc-finger domain of isoforms A and B of WT1 with high affinity and specificity. A mutation in the binding site that disrupted this interaction partially removed the inhibition of ODC promoter activity by WT1, as did mutation of the two E-box sequences in intron I of the ODC gene. Simultaneous mutation of the WT1-binding motif and the two E-boxes completely abolished inhibition by WT1 of ODC promoter activity. These results, taken together, implicate the ODC gene as a downstream target of the tumor suppressor WT1.

Transcription factor ZBP-89 regulates the activity of the ornithine decarboxylase promoter.

Appropriate cellular levels of polyamines are required for cell growth and differentiation. Ornithine decarboxylase is a key regulatory enzyme in the biosynthesis of polyamines, and precise regulation of the expression of this enzyme is required, according to cellular growth state. A variety of mitogens increase the level of ornithine decarboxylase activity, and, in most cases, this elevation is due to increased levels of mRNA. A GC box in the proximal promoter of the ornithine decarboxylase gene is required for basal and induced transcriptional activity, and two proteins, Sp1 and NF-ODC1, bind to this region in a mutually exclusive manner. Using a yeast one-hybrid screening method, ZBP-89, a DNA-binding protein, was identified as a candidate for the protein responsible for NF-ODC1 binding activity. Three lines of evidence verified this identification; ZBP-89 copurified with NF-ODC1 binding activity, ZBP-89 antibodies specifically abolished NF-ODC1 binding to the GC box, and binding affinities of 12 different double-stranded oligonucleotides were indistinguishable between NF-ODC1, in nuclear extract, and in vitro translated ZBP-89. ZBP-89 inhibited the activation of the ornithine decarboxylase promoter by Sp1 in Schneider's Drosophila line 2, consistent with properties previously attributed to NF-ODC1.

Expression of apolipoprotein E mRNA in the epithelium and interstitium of the testis and the epididymis.

Apolipoprotein E (apo E) is an important constituent of plasma lipoproteins and is believed to be involved in the regulation of lipid transport and distribution between tissues. The production of this apolipoprotein in extra-hepatic tissues such as the testis and epididymis could facilitate specific local functions. Apo E mRNA was detected in testis, epididymis, seminal vesicles, and prostate. In the epididymis, apo E was detected using in situ hybridization in epithelial cells and in some cells in the interstitium throughout the organ (i.e., caput, corpus, and cauda). Northern blot analysis showed that apo E mRNA is present in Sertoli cells and germ cells, but not peritubular myoid cells. Interstitial cells of the testis displayed the most intense signal for apo E message using in situ hybridization. Messenger RNA for apo E was also detected in the interstitium of rat testes at 3 and 6 days after animals were treated with ethylene dimethanesulfonate (EDS) to eliminate Leydig cells. Thus, in addition to Leydig cells, other cell types within the interstitium are capable of producing apo E message. Levels of testicular apo E mRNA increased between 30 and 60 days pc during which the germ cell population is increasing. As determined by northern blot analysis of RNA from stage synchronized testes, the levels of apo E mRNA fluctuate in relation to the cycle of the seminiferous epithelium. The cells responsible for this stage-specific variation in message could not be identified by in situ hybridization. Apolipoprotein Al (apo Al) mRNA was also found to be expressed in the epididymis but not in the testis of adult rats. The role of apolipoproteins in spermatogenesis and sperm maturation has not been elucidated. The results of this study demonstrate the specific tissues and cells types which play a role in the production and possible regulation of apo E mRNA in the male reproductive tract. These data will help in the elucidation of the function of apo E in spermatogenesis and sperm maturation.

Activity and form of sulfated glycoprotein 2 (clusterin) from cultured Sertoli cells, testis, and epididymis of the rat.

Sulfated glycoprotein 2 (SGP 2) is a 73-kDa highly glycosylated disulfide-linked heterodimer. It is a major secreted protein of Sertoli cells, is found in high abundance within the seminiferous tubule fluid (STF) and epididymal fluid (EPF), and can be found on the surface of spermatozoa. Due to its high abundance and location it is believed to play a major role in the development of spermatozoa; however, its specific function(s) within the reproductive tract is not known. Purified and renatured SGP 2 were found to have the ability to inhibit complement activity with a mean concentration of 66 mg/ml for 50% inhibition. Extraction of epididymal sperm with various reagents showed that a major fraction of the SGP 2 in EPF was free or was loosely associated with the spermatozoa whereas a smaller fraction was more tightly associated and disruption of the lipid bilayer was required for its complete removal. Ultracentrifugation techniques and gel permeation chromatography revealed that SGP 2 in plasma, STF, and EPF formed complexes with other proteins and/or lipids but was not specifically associated with the apolipoprotein-like particles containing apolipoprotein A1 (apo A1).