ASCORBATE-SENSING uORF CONTROLS GDP-L-GALACTOSE PHOSPHORYLASE (GGP) mRNA TRANSLATION

According to the scanning model of eukaryotic translation initiation, ribosomes bind near the 5’ cap of an mRNA and then initiate translation at the first start codon they encounter as they progress down the mRNA. Upstream open reading frames (uORFs) can regulate translation initiation at downstream start codons. Recently, a uORF in the GDP-L-galactose phosphorylase (GGP) mRNA was reported to mediate autoregulation of ascorbate, or Vitamin C, production. Ascorbate is an essential enzymatic cofactor and antioxidant in plants and animals. Ascorbate concentrations are tightly regulated in plants through post-transcriptional repression of GGP, the major enzyme controlling ascorbate biosynthesis. Key attributes of the noncanonical GGP uORF include: (1) initiation at either a near-cognate start codon or an AUG codon in poor context; (2) it encodes a 32-65 residue peptide containing conserved motifs; and (3) ribosome profiling indicates the presence of an elongation pause at the uORF C-terminus. These attributes suggest that the GGP uORF is a novel upstream Conserved Coding region (uCC), a new paradigm of translational control involving a uORF whose initiation at a weak start site is triggered by queuing of scanning ribosomes behind an elongating ribosome paused at a conserved amino acid motif within the uCC. We hypothesize that ascorbate promotes pausing of translation elongation in the uORF to enhance uORF translation and to repress translation of GGP. To assess ascorbate mediated GGP repression by the uORF, we examined reporter constructs in mammalian cell lines and in rabbit reticulocyte lysate in vitro translation systems. Alignment of 20 algae species’ GGP uORFs shows absolute conservation of five amino acid motifs. Consistent with the GGP uORF being a uCC, conservation of all five motifs and their spacing is essential for regulation of GGP mRNA translation by ascorbate in vivo and in vitro, and the elongation pause is specific to ascorbate. Ribosome profiling from Chlamydomonas reinhardtii and Solanum lycopersicum showed an elongation pause at the C-terminal glycine repeat. In support of the queuing model, reducing ribosome loading by inserting a cap-proximal stem-loop in the GGP mRNA abolished ascorbate specific regulation of GGP expression. Taken together, these data indicate that the GGP uORF is a uCC. We propose that ascorbate interacts with conserved residues of the uCC nascent peptide within the ribosome exit tunnel, causing an elongation pause that regulates GGP mRNA translation