Regulated Translation Termination at the Upstream Open Reading Frame in S-Adenosylmethionine Decarboxylase mRNA

Abstract

The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a cis-acting element that confers feedback control by cellular polyamines on translation of this message. Recent studies demonstrated that elevated polyamines inhibit synthesis of the peptide encoded by the uORF by stabilizing a ribosome paused in the vicinity of the termination codon. These studies suggested that polyamines act at the termination step of uORF translation. In this paper, we demonstrate that elevated polyamines stabilize an intermediate in the termination process, the complete nascent peptide linked to the tRNA that decodes the final codon. The peptidyl-tRNA molecule is found associated with the ribosome fraction, and decay of this molecule correlated with release of the paused ribosome from the message. Furthermore, the stability of this complex is influenced by the same parameters that influence regulation by the uORF in vivo, namely the concentration of polyamines and the sequence of the uORF-encoded peptide. These results suggest that the regulated step in uORF translation is after formation of the peptidyl-tRNA molecule but before hydrolysis of the peptidyl-tRNA bond. This regulation may involve an interaction between the peptide, polyamines, and a target in the translational apparatus. S-Adenosylmethionine decarboxylase (AdoMetDC)1 is a key regulated enzyme in the pathway of polyamine biosynthesis. AdoMetDC catalyzes the decarboxylation of S-adenosylmethionine, generating the n-propylamine donor for the conversion of putrescine to spermidine and spermidine to spermine. These polyamines are required for cell growth and have been implicated in many cellular processes including DNA replication, transcription, and translation (1, 2).Spermidine and spermine feedback inhibit AdoMetDC translation by acting through the peptide produced from a small upstream open reading frame (uORF) located in the leader of the AdoMetDC transcript (3-5). The peptide product of the uORF is six amino acids long and has the sequence MAGDIS. This peptide seems to act in cis on the mRNA that produced it, and features of the carboxyl-terminal sequence are of particular importance (4, 6). Saturation mutagenesis of the last three codons of the uORF demonstrated that only aspartic acid is tolerated in the fourth position and only the isoleucine homologue, valine, can substitute in the fifth position (4). The specific structural requirements of the fourth and fifth amino acids are demonstrated by the fact that glutamic acid does not fully substitute for aspartic acid, and substitution of isoleucine with another homologue, leucine, completely abolishes regulation (4).These studies imply a precise interaction between the MAG-DIS peptide and its regulatory target. In one model of AdoMetDC regulation, polyamines could act to modulate an interaction of the fourth and fifth amino acids of the peptide product of the AdoMetDC uORF with a component of the translational apparatus, such as the peptidyltransferase center of the ribosome or ...

Inhibition of Translation Termination Mediated by an Interaction of Eukaryotic Release Factor 1 with a Nascent Peptidyl-tRNA

“…Our results raise the question of whether release factors play a role in other uORFmediated ribosomal stalling events. The S-adenosylmethionine decarboxylase uORF codes for a sequence-dependent polyamine-responsive peptide that, like uORF2, causes ribosomal stalling specifically at the termination codon and results in accumulation of the uORF peptidyl-tRNA (30,40). While the critical sequences of this uORF do not include the carboxyterminal residue, the penultimate and antepenultimate residues have been implicated (35).…”

RNAi Suppression of Arogenate Dehydratase1 Reveals That Phenylalanine Is Synthesized Predominantly via the Arogenate Pathway in Petunia Petals

“…Currently, the nature of this posttranscriptional regulation of the shikimate pathway, which can occur at the level of translation (Wang and Sachs, 1997;Raney et al, 2002), protein modification (Huber and Huber, 1996;Savage and Ohlrogge, 1999;Tetlow et al, 2004;Uhrig et al, 2008), and/or enzyme activity (Ghosh and Preiss, 1966;Gilchris et al, 1972;Chollet et al, 1996), is unknown. Exogenously supplied shikimate clearly bypasses this negative regulation and leads to elevated accumulation of arogenate in transgenic petals (Figure 10), suggesting that the regulation occurs upstream of shikimate.…”

Polyamine-Regulated Translation of Spermidine/Spermine-N1-Acetyltransferase

“…Because so many processes are affected, levels are maintained within a relatively narrow range by shifts in anabolism/catabolism and import/export (1, 46). Manipulation of polyamine metabolism has been an anticancer strategy, with pool depletion in tumor cells used as a surrogate marker of efficacy (21,34,38,48).Many mechanisms contribute to control of eukaryotic polyamine metabolic enzymes (15,41,43,50). Ornithine decarboxylase (ODC), the rate-limiting anabolic enzyme, is regulated allosterically, at transcription, at translation, and by "ODC antizyme," a protein that binds to ODC monomers, thereby blocking homodimerization required for activity and accelerating monomer degradation (41).…”