The zipper model of translational control: a small upstream ORF is the switch that controls structural remodeling of an mRNA leader

Transport of the essential amino acids arginine and lysine is critical for the survival of mammalian cells. The adaptive response to nutritional stress involves increased translation of the arginine/lysine transporter (cat-1) mRNA via an internal ribosome entry site (IRES) within the mRNA leader. Induction of cat-1 IRES activity requires both translation of a small upstream open reading frame (uORF) within the IRES and phosphorylation of the translation initiation factor eIF2alpha. We show here that translation of the upstream ORF unfolds an inhibitory structure in the mRNA leader, eliciting a conformational change that yields an active IRES. The IRES, whose activity is induced by amino acid starvation, is created by RNA-RNA interactions between the 5' end of the leader and downstream sequences. This study suggests that the structure of the IRES is dynamic and regulation of this RNA structure is a mechanism of translational control.     

A bicistronic Epstein-Barr virus mRNA encodes two nuclear proteins in latently infected, growth-transformed lymphocytes.

EBNA2 is a nuclear protein expressed in all cells latently infected with and growth transformed by Epstein-Barr virus (EBV) infection (K. Hennessy and E. Kieff, Science 227:1230-1240, 1985). The nucleotide sequence of the EBNA2 mRNA (J. Sample, M. Hummel, D. Braun, M. Birkenbach, and E. Kieff, Proc. Natl. Acad. Sci. USA 83:5096-5100, 1986) revealed that it begins with a 924-base open reading frame that has an unusual potential translational initiation site (CAAATGG). This open reading frame is followed by 138 nucleotides with only one highly unlikely translational initiation site (TACATGC), which would translate a pentapeptide before the next stop codon. The last part of the mRNA is the open reading frame which encodes EBNA2. In this paper, we demonstrate that the 924-base open reading frame translates a 40-kilodalton protein in vitro or in murine cells transfected with the EBNA2 cDNA under control of the murine leukemia virus long terminal repeat. A protein of identical size was detected in EBV-transformed, latently infected human lymphocyte nuclei by using antibody specific for the leader open reading frame expressed in bacteria. Therefore, this is a rare example of a mRNA which translates two proteins from nonoverlapping open reading frames. Since the protein encoded by the leader of the EBNA mRNA is expressed in all nuclei of a latently infected cell line, it was designated EBNA-LP. EBNA-LP localizes to small intranuclear particles and differs in this respect from EBNA1, EBNA2, or EBNA3. EBNA-LP is not expressed in an EBV-transformed marmoset lymphocyte cell (B95-8) or in one EBV-infected Burkitt tumor cell line (Raji) but is expressed in three other Burkitt tumor cell lines (Namalwa, P3HR-1, and Daudi).     

Regulation of rat ornithine decarboxylase mRNA translation by its 5'-untranslated region

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is a highly inducible protein whose expression involves a complex and variable array of regulatory mechanisms. We investigated the influence of the 5'-untranslated region (5'UTR) of the rat ODC mRNA on translation of the mRNA in a cell-free system and in cultured mammalian cells. ODC mRNA containing the full-length 5'UTR was translated in reticulocyte lysates at approximately 5% of the rate of mRNA containing no ODC 5' leader sequences. The complete 5'UTR inhibited expression of a heterologous gene product, human growth hormone, to the same extent in cultured mammalian cells. Furthermore, the 5'-most 130 bases of the rat ODC 5'UTR, a conserved G/C-rich region predicted to form a stable stem-loop structure (delta G = -68 kcal/mol), repressed translation to the same extent as the entire 5'UTR, both in the lysates and in intact cells. The 3'-most 160 bases of the 5'UTR, containing a small upstream open reading frame, decreased expression by 50-65% both in vitro and in intact cells, compared with controls lacking any ODC 5'UTR sequences. Mutation of the initiation codon AUG beginning this upstream open reading frame to GCG restored expression to rates equivalent to those seen in constructions containing no ODC 5'UTR sequences. We conclude that the rat ODC mRNA 5'UTR can inhibit translation of ODC mRNA both in vitro and in vivo, and that the predicted stem-loop structure at the 5' end of the 5'UTR is both necessary and sufficient for this inhibition.     

Role of an upstream open reading frame in the translation of polycistronic mRNAs in plant cells.

The influence of an upstream small open reading frame (URF) on the translation of two consecutive coding regions on an eukaryotic mRNA was studied. The cis effects of leader length, URF length, the sequences of the URF and neighboring regions, and the trans effects of the Cauliflower mosaic virus transactivator (TAV) were analyzed. Translation efficiency of the immediate downstream open reading frame (ORF) decreased with increasing URF length. Short URFs did not drastically inhibit translation of immediate downstream ORFs but supported far downstream translation in the presence of TAV. In the latter case, the optimal URF length was 30 codons.     

The positive regulatory function of the 5''-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function.