Poster abstracts
Poster number 13 submitted by Pooja Gangras
Proximal 3'UTR introns elicit EJC-dependent NMD during zebrafish embryonic development
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:
Post-transcriptional control of gene expression is essential for proper development, and is achieved largely by RNA-binding proteins. One such protein complex, the Exon Junction Complex (EJC), is deposited 24 nts upstream of exon-exon junctions during pre-mRNA splicing. The EJC influences many aspects of post-transcriptional regulation, including Nonsense Mediated mRNA Decay (NMD). NMD is a surveillance system that degrades aberrant mRNAs and non-aberrant mRNAs containing ‘NMD-inducing features’ such as 3’UTR introns (3’UIs). Post-splicing, a 3’UI leads to an EJC bound downstream of the stop codon – if the distance between the two is ≥50 nts, the mRNA is targeted for NMD by the NMD-regulator Upf1. To study EJC function during development, we generated zebrafish mutants in EJC core protein genes rbm8a and magoh. Homozygous rbm8a and magoh mutants are paralyzed and have muscle and neural defects. As expected, RNA profiling reveals that annotated aberrant and natural NMD targets are significantly upregulated in EJC mutants. Surprisingly, some upregulated natural transcripts contain a conserved proximal 3’UI (<50 nts downstream of the stop codon). These ‘proximal 3’UI-containing NMD targets’ are similarly upregulated in Upf1-deficient and NMD inhibitor-treated embryos, suggesting that this subset of rbm8a- and magoh-regulated transcripts is regulated via NMD. The same trend is observed in Upf1-deficient mammalian cells. One proximal 3’UI-containing NMD target transcript encodes Foxo3b, which is known to play a role in autophagy, muscle atrophy and Wnt signaling. Consistent with our hypothesis that rbm8a and magoh mutant defects are due, at least in part, to increased Foxo3b activity, we show that heterozygous knockout of foxo3b in EJC mutants leads to partial phenotypic rescue. Our findings show that proximal 3'UTR introns are a new, atypical NMD-inducing feature that may be critical for regulating gene expression during embryogenesis.
Keywords: EJC-dependent NMD, zebrafish, development