uORFdb - a comprehensive literature database on eukaryotic uORF biology

To allow convenient and targeted access to the complex field of eukaryotic uORF biology, we generated uORFdb, publicly available at http://www.compgen.uni-muenster.de/tools/uorfdb, to serve as a comprehensive literature database on eukaryotic uORF biology. The uORFdb was manually curated from all uORF-related literature listed at the PubMed database. It categorizes individual publications by a variety of denominators including taxon, gene and type of study. Furthermore, the database can be filtered for multiple structural and functional uORF-related properties.

http://www.compgen.uni-muenster.de/tools/uorfdb

CEBPBΔuORF mice – a genetic model for uORF-mediated translational control

In cooperation with the group of Achim Leutz (MDC, Berlin) we showed that mice deficient for the CCAAT/enhancer-binding protein beta (CEBPB) uORF initiation codon failed to initiate translation of the autoantagonistic LIP (liver inhibitory protein) CEBPB isoform. CEBPBΔuORF mice showed hyperactivation of acute-phase response genes, persistent repression of E2F-regulated genes, delayed and blunted S-phase entry of hepatocytes after partial hepatectomy, and impaired osteoclast differentiation. This study provided initial experimental evidence that a point mutation in a uORF start site resulted in physiological perturbations in mice, suggesting that similar genetic defects in other genes may promote pathogenic overexpression of uORF-controlled proteins.

Genetic ablation of cis-regulatory translational control in mice

Three CEBPB protein isoforms LAP* (Liver Activating Protein*, 38 kD), LAP (35 kD) and LIP (Liver Inhibitory Protein, 20 kD) are translated from consecutive in-frame initiation codons in the same transcript. Induction of LIP in C/EBPBWT livers upon partial hepatectomy is abolished in C/EBPBΔuORF animals. “h” indicates hours after surgery.

uORF-mediated translational control of proto-oncogene expression

We observed constitutive uORF-mediated translational repression in a number of human tyrosine kinases and other proto-oncoproteins, hinting towards a mechanism of enhanced proto-oncogene expression through loss-of-function uORF mutations.  

A) Graphic representation of the luciferase reporter construct. The plasmid allows the insertion of a complete TLS including the endogenous mAUG initiation codon plus the surrounding Kozak sequence including base +4 (N) to retain the gene specific initiat

A) Graphic representation of the luciferase reporter construct. The plasmid allows the insertion of a complete TLS including the endogenous mAUG initiation codon plus the surrounding Kozak sequence including base +4 (N) to retain the gene specific initiation context. For functional experiments, the wild type (wt) uORF initiation codon was deleted by the insertion of a point mutation that creates a UUG codon instead (∆uORF).
B) Schematic representation of the position and length of uORFs within selected TLSs. Conservation of the uAUG among human and mouse (check mark) and among a total of nine vertebrate species (human, rhesus, mouse, rat, cow, dog, elephant, chicken, zebrafish) is depicted. The Kozak context is indicated as strong (++, a purine base at -3 and a guanine base at +4), intermediate (+, one of the core Kozak bases matches), or weak (-, no core Kozak base matches).
C) Bar graph showing the relative reporter activity in the presence of wt uORF and ∆uORF containing TLSs of indicated proteins. For each construct, Firefly luciferase signals were normalized to Renilla luciferase internal control signals.
D) Bar graph indicating the relative luciferase mRNA levels of wt uORF and ∆uORF reporter constructs for indicated TLSs.
Error bars represent the SEM of three independent experiments. Asterisks indicate statistical significance (**p<0.01, *p<0.05).

Current work

Based on these findings, we recently developed a screening approach combining PCR amplification and deep sequencing to simultaneously investigate a high number of uORF start site regions in various cancer samples of different entities. We screened 308 human malignancies for loss-of-uAUG mutations in 404 uORF initiation sites of 132 potential proto-oncogenes. Our screen identified novel loss-of-uAUG and uKozak-affecting mutations in various types of cancer and suggest that genetic defects in uORF-mediated regulation may contribute to malignant transformation of human cells. Current efforts focus on the characterizing patho-physiological mechanisms driven by the newly identified uORF-related mutations in various types of human cancer.

 
 
 
 

Kontakt

Universitätsklinikum Münster Medizinische Klinik A
AG Translationale Regulation der Genexpression

Leitung
Dr.med. Dr.rer.nat. Klaus Wethmar

Albert-Schweitzer-Campus 1, Gebäude D3,

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