Poster abstracts
Poster number 18 submitted by Pooja Gangras
EJC acts via NMD to manipulate levels of key developmental regulators
Pooja Gangras (Department of Molecular Genetics), Thomas L. Gallagher (Department of Molecular Genetics), Kiel T. Tietz (Department of Molecular Genetics), Natalie C. Deans (Department of Molecular Genetics), Sharon L. Amacher (Department of Molecular Genetics), Guramrit Singh (Department of Molecular Genetics)
Abstract:
Developmental gene expression is regulated at transcriptional and post-transcriptional levels. One post-transcriptional regulatory mechanism is Nonsense Mediated mRNA Decay (NMD) which was originally described as a surveillance system to degrade aberrant mRNAs. It is now appreciated that NMD also degrades non-aberrant mRNAs containing ‘NMD-inducing features’ such as 3’ UTR introns. Post-splicing, an mRNA with a 3’ UTR intron has an Exon Junction Complex (EJC) bound downstream of the stop codon – if the distance between the two is ≥ 50 nts, the mRNA is recognized by the key NMD-regulator Upf1 and targeted for NMD. To study EJC function during development, we generated zebrafish mutants in EJC core proteins Rbm8a and Magoh. Homozygous rbm8a and magoh mutants are paralyzed and have disorganized myofibrils and stunted motoneuron axons. As expected, RNA profiling reveals that annotated NMD targets are significantly upregulated in EJC mutants; ten of these show the same trend in Upf1-deficient embryos and some contain known NMD-inducing features, suggesting that this subset may be regulated via NMD. Among the other upregulated transcripts, three contain a conserved 3’ UTR intron < 50 nts downstream of the stop codon. One of these encodes Foxo3b, a transcription factor that functions to inhibit Wnt signaling during development. We hypothesized that increased Foxo3b in EJC mutants might decrease Wnt signaling below a critical threshold. To test this, we treated EJC mutants with a Wnt agonist BIO, and preliminary results show dramatic rescue of mutant morphological defects. We hypothesize that a distal EJC (≥ 50 nts downstream of the stop codon) directs rapid mRNA turnover while a more proximal EJC (< 50 nts) creates a sub-optimal NMD target that decays more gradually, allowing finer tuning of expression levels. To test this hypothesis, I will conduct foxo3b mini-gene experiments and probe our datasets to discover additional NMD targets and atypical NMD-inducing features.
Keywords: Exon Junction Complex, NMD, development