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The inhibitory upstream open reading frame from mammalian S-adenosylmethionine decarboxylase mRNA has a strict
1Department of Biochemistry, University of Washington, Seattle, Washington 98195-7350, USA.
Abstract:
The upstream open reading frame (uORF) in the 5' leader of the mammalian mRNA encoding S-adenosylmethionine decarboxylase (AdoMetDC) serves as a negative regulatory element by suppressing translation of the associated downstream cistron. Certain changes in the amino acid sequence of the hexapeptide (sequence MAGDIS) encoded by the uORF destroy suppressive activity, implying specific interaction with a cellular target. In this paper, we examine the extent of alterations that can be tolerated in this uORF. The mammalian AdoMetDC uORF inhibits downstream translation when placed into the 5' leader of a yeast mRNA with characteristics resembling those in mammalian cells, suggesting that the encoded peptide has a similar target across species. Using yeast for the initial screen, we tested the specificity of the critical three codons at the 3' end of the uORF by saturation mutagenesis. Altered uORFs selected from the primary yeast screen were then retested in mammalian cells. The requirements at codons 4 and 5 were quite stringent; only aspartic acid at codon 4 yielded a fully suppressive peptide, and only valine could substitute productively for isoleucine at codon 5. The specificity at codon 6 was much looser, with many substitutions retaining suppressive activity in both yeast and mammalian cells.
Insights
Altering the S-adenosylmethionine decarboxylase (AdoMetDC) mRNA's upstream open reading frame (uORF) hexapeptide sequence impacts translation suppression. Specific amino acid changes at codons 4 and 5 are critical for maintaining this regulatory function across species.
Area of Science:
- Molecular Biology
- Gene Regulation
- Biochemistry
Background:
- The upstream open reading frame (uORF) in the S-adenosylmethionine decarboxylase (AdoMetDC) mRNA acts as a translational repressor.
- Changes in the uORF-encoded hexapeptide (MAGDIS) can abolish this suppressive activity, suggesting specific molecular interactions.
Purpose of the Study:
- To investigate the tolerance for sequence alterations within the mammalian AdoMetDC uORF.
- To determine the specificity of the encoded hexapeptide for its cellular target.
- To assess cross-species conservation of the uORF's regulatory mechanism.
Main Methods:
- Utilized a yeast mRNA system to screen for alterations in the AdoMetDC uORF that affect translational suppression.
- Employed saturation mutagenesis at the 3' end of the uORF in yeast.
- Re-tested selected altered uORFs in mammalian cells to validate findings.
Main Results:
- The mammalian AdoMetDC uORF retained suppressive activity when transferred to yeast, indicating conserved targets.
- Codon 4 was highly specific, with only aspartic acid maintaining full suppressive activity.
- Codon 5 showed some flexibility, allowing valine to substitute for isoleucine effectively.
- Codon 6 exhibited broad tolerance for amino acid substitutions without losing suppressive function in both yeast and mammalian cells.
Conclusions:
- The AdoMetDC uORF's translational regulatory function is conserved across yeast and mammalian cells.
- Specific amino acid residues within the uORF-encoded peptide are crucial for interacting with cellular targets and mediating translational repression.
- The study provides insights into the sequence requirements for uORF-mediated gene regulation.