Abstract
S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the pathway of polyamine biosynthesis. The cellular levels of the polyamines specifically regulate AdoMetDC translation through the 5'-leader of the mRNA, which contains a small upstream open reading frame (uORF) 14 nucleotides from the cap. Mutating the initiation codon of the uORF, which encodes a peptide product with the sequence MAGDIS, abolished regulation. In addition, the uORF is sufficient, by itself, to provide polyamine regulation when inserted into the 5'-leader of the human growth hormone mRNA. Changing the amino acid sequence at the carboxyl terminus of the peptide product of the uORF abolished polyamine regulation. In contrast, altering the nucleotide sequence of the uORF at degenerate positions, without changing the amino acid sequence of the peptide, did not affect regulation. Extending the distance between cap and uORF, thereby changing the rate of initiation at the initiator AUG of the uORF, did not alter polyamine regulation. When the uORF was extended so as to overlap, out of frame, the downstream major cistron, polyamine regulation was abolished. We propose that polyamines do not modulate the rate of recognition of the uORF but rather regulate interaction of the peptide product of the uORF with its target.
Highlights
Polyamines are a group of low molecular weight compounds necessary for the optimal growth of all cells, eukaryotic and prokaryotic (for reviews, see Marton and Morris (1987) and Janne et al (1991))
Several studies have focused on translational control of AdoMetDC by polyamines, using polyamine-depleting drugs, such as ␣-difluoromethylornithine (DFMO), a potent irreversible inhibitor of the first enzyme in polyamine biosynthesis, ornithine decarboxylase (for review, see Marton and Pegg (1995))
Intracellular polyamine levels influence the translation of AdoMetDC mRNA in vivo, and the major effect seems to be at the initiation step (White et al, 1990)
Summary
Chimeric Constructs—Chimeras between AdoMetDC and human growth hormone (hGH) were described in previous publications (Hill and Morris, 1992, 1993; Ruan et al, 1994). In experiments in which polyamines were depleted, a final concentration of 5 mM DFMO was added in the media. Triplicate cultures at each time point were washed twice with culture medium lacking methionine, and medium containing [35S]methionine was added (600 l of medium containing 10% fetal bovine serum with 2 Ci of [35S]Met per 35-mm plate). The cell pellet was washed with 10% trichloroacetic acid twice, once with an ethanol:ether mixture (1:1), and once with ether. For cells depleted of polyamines, a final concentration of 5 mM DFMO was added at plating and was present throughout the transfection. In experiments where polyamines were restored to depleted cells, a final concentration of 10 M spermidine (with 1 mM aminoguanidine) was added at the time of transfection
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
