Upstream open reading frames: molecular switches in (patho)physiology

Abstract

Conserved upstream open reading frames (uORFs) are found within many eukaryotic transcripts and are known to regulate protein translation. Evidence from genetic and bioinformatic studies implicates disturbed uORF-mediated translational control in the etiology of human diseases. A genetic mouse model has recently provided proof-of-principle support for the physiological relevance of uORF-mediated translational control in mammals. The targeted disruption of the uORF initiation codon within the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) gene resulted in deregulated C/EBPβ protein isoform expression, associated with defective liver regeneration and impaired osteoclast differentiation. The high prevalence of uORFs in the human transcriptome suggests that intensified search for mutations within 5' RNA leader regions may reveal a multitude of alterations affecting uORFs, causing pathogenic deregulation of protein expression.

Figure 1

Variables affecting the degree of uORF-mediated MCS repression. The enhancement of MCS repression correlates with increasing m⁷G to uORF distance, uORF to MCS proximity, uORF length, number and conservation among species, and an increasingly favorable uORF initiation context. *These features apply to individual transcripts but have not been validated in a bioinformatic survey , , , . m7G, 5′ mRNA cap structure; uORF, upstream open reading frame; MCS, main coding sequence.

Figure 2

Transcripts and protein isoforms of C/EBPα and β transcription factors. Three N-terminally distinct protein isoforms (colored bars) are translated from subsequent in-frame initiation codons (black arrows) within the C/EBPα and β transcripts (open bars). Small uORFs (blue) preceding the initiation codons of C/EBPα-p42 and C/EBPβ-LAP regulate the balanced expression of long and truncated protein isoforms. The C/EBPα and β isoforms contain N-terminal trans-activating (green) and regulatory (red) domains, as well as highly conserved C-terminal basic (orange) and leucine zipper (purple) domains. The positions and sizes of indicated domains are derived from published data , –. C/EBP, CCAAT enhancer binding protein; ext, extended; LAP, liver activating protein; LIP, liver inhibitory protein.

Figure 3

C/EBPα and β isoform expression ratios affect cellular proliferation and differentiation, and are modulated in response to mTOR signaling. A: Several examples of how the C/EBP isoform ratio affects cellular differentiation are illustrated, specifically how an increase in the short isoforms p30 and LIP disrupts proper differentiation. For example, p30 and LIP are overexpressed in several human cancers, including AML and breast cancer, respectively. The truncated isoforms are sufficient to induce lineage commitment of proliferative progenitor cells; however, they are not capable of blocking the cell cycle (indicated with the circular arrow) and inducing terminal differentiation and maturation. * In these cases, similar isoform specific functions have been described for both, C/EBPα and β. B: Environmental signals enhance (green) or repress (red) mTOR kinase activity, resulting in changes in global translational conditions. Changes in the translational status have been shown to affect uORF translation, resulting in changes in C/EBP protein isoform balance. In a good translational status, the C/EBPα and β uORFs may be more frequently translated, shifting the isoform expression ratio toward the truncated C/EBPα (p30) and β (LIP) isoforms (green).

Figure 4

Validated and hypothetical uORFs in C/EBP transcription factors. C/EBPα and β transcripts of human (homo), cow (bos), and mouse (mus) contain experimentally validated uORFs (gray background color) terminating 7 and 4 nucleotides in front of the p42 and LAP initiation codon, respectively. The most abundant C/EBPɛ transcript variant contains three subsequent hypothetical uORF initiation codons (uORF^hyp), followed by in-frame termination codons upstream (homo) or downstream (bos and mus) of the p30 start site. The rat C/EBPɛ sequence is not displayed, as it is 100% homologous to the mouse sequence shown in this alignment. Initiation codons of protein isoforms are highlighted by green background color, initiation and termination codons of uORFs and uORFs^hyp are in red bold face, favorable residues of the core Kozak context (residues at −3 or +4) are underlined. * This uORF^hyp initiation codon may be nonfunctional, as its presence did not prevent deregulated C/EBPβ isoform expression when the uORF AUG codon (−34) was mutated to a non-functional UUG codon , .

Figure 5

How uORF mutations may drive malignant transformation. Mutations (lightning arrows) that eliminate uORFs may activate the translation of transforming proto-oncogenes. Mutations that create uORFs in front of tumor suppressor genes may decrease translation of the encoded protective protein (as shown for CDKN2A 84). Either way, uORF-affecting mutations may result in malignant transformation of cells.