Upstream ORFs and Regulation of Translation

Protein expression can be rapidly and responsively regulated at the level of translation. Translational regulation commonly involves trans-acting factors such as miRNA complexes or RNA-binding proteins, specifically binding cis-regulatory sequences. A study from last year – Medenbach et al. – has demonstrated an interesting mechanism in which translation of a transcript is repressed by the initiation of translation at a short upstream open reading frame (uORF).

Sexual organisms need a mechanism to balance the levels of transcription from sex chromosomes between sexes. In mammals this ‘dosage compensation’ is achieved by inactivating one of the X chromosomes in females; Drosophila instead hypertranscribes the single X chromosomes of male flies. In females, hypertranscription is prevented by the translational silencing of male-specific lethal (msl)-2 mRNA by a key sex determination factor, the RNA-binding protein Sex-lethal (SXL).

SXL exerts translational control on msl-2 transcripts by two distinct mechanisms. Binding in the 3’UTR, in conjunction with a co-repressor, it blocks the recruitment of the ribosomal pre-initiation complex to the 5’UTR. Any pre-initiation complexes that escape this control are then challenged by a failsafe mechanism; SXL bound to the 5’UTR causes destabilisation of the small ribosomal subunit upstream of the SXL-binding site.

Medenbach et al. analysed the mechanisms of this 5’UTR SXL-mediated translational control, using constructs in which altered msl-2 5’UTRs drove expression of a reporter gene in a cell-free system. The msl-2 5’UTR contains three small upstream ORFs. Mutations preventing initiation in the first two of these uORFs did not impair translational control, but when the third uAUG was mutated the reporter construct was translationally de-repressed. This effect was dependent on a SXL-binding site slightly downstream of the uORF, mutation of which abrogated the derepression. The repression was dependent on the presence of SXL. By itself the uORF reduced translation of the downstream reading frame two fold, but in the presence of SXL, downstream translation was repressed more than 14 fold.

The uORF encodes a di-peptide (Methionine-Threonine). By swapping the threonine-encoding codon and the subsequent stop codon, and by deleting the stop codon, the researchers showed that translational repression required uORF initiation and not translational elongation or termination.

Medenbach et al. went on to investigate just how widespread this mechanism of translational control may be. Computationally scanning the Drosophila transcriptome, they found that 58% of 5’UTRs contain one or more upstream initiation codons, whilst 4.3% contain putative SXL binding sites. 268 mRNAs (1.3%) contain both a uORF and a SXL-binding motif at the appropriate distance from one another to make them candidates for a similar mode of SXL-mediated translational repression. They then tested 12 of these candidate 5’UTRs in their reporter assay, and found that 6 of them did indeed mediate SXL-dependent translational repression. They also demonstrated that the regulatory cassette consisting of the uORF, the intervening 21nt, and the SXL-binding site was capable of mediating repression when inserted into the 5’UTR of an unrelated gene.

uORFs have previously been shown to regulate translation of the main reading fame in other contexts. The authors reference two examples using different mechanisms; a system involving termination and reinitiation from 4uORFs in a budding yeast transcript that doesn’t require any trans-acing factors; and another fungal mechanism in which ribosomal stalling at the termination codon of an uORF causes the mRNA to be degraded. In contrast, the translation regulation system in the msl-2 5’UTR utilises a binary module requiring only translational initiation at the uORF and binding of SXL. How exactly this works with respect to the ribosomal pre-initiation complex is not yet clear. Recognition of an initiation codon triggers a conformational change in this complex from a scanning ‘open’ structure to a closed conformation, followed by subunit joining to make a complete translationally competent ribosome. The authors found that SXL interacts with two subunits of the elF3 initiation factor, but weren’t able to prove the importance of these interactions for translational repression.

Medenbach et al’s computational scan of the Drosophila genome showed that the use of this binary module is an important mechanism by which SXL regulates the expression of many proteins. Similar uORF modules may be utilised by other RNA binding proteins, but it’s also possible that other uORFs function in quite different ways, as suggested by the examples from fungi. What’s beyond doubt, seeing as more than half of mammalian and Drosophila genes contain them is that uORFs are an important factor in translational regulation.

Medenbach J, Seiler M, & Hentze MW (2011). Translational control via protein-regulated upstream open reading frames. Cell, 145 (6), 902-13 PMID: 21663794

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