The Inhibitory Upstream Open Reading Frame from Mammalian S-Adenosylmethionine
Decarboxylase mRNA Has a Strict Sequence Specificity in Critical Positions.
Gregory J. Mize, Hangjun Ruan, Justin Low and David R. Morris
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.