Poster abstracts
Poster number 9 submitted by Pooja Gangras
Proximal 3'UTR introns elicit EJC-dependent NMD during zebrafish development.
Pooja Gangras (Department of Molecular Genetics), Thomas L. Gallagher (Department of Molecular Genetics), Robert D. Patton (Department of Molecular Genetics), Zhongxia Yi (Department of Molecular Genetics), Ralf Bundschuh (Department of Physics, Department of Chemistry and Biochemistry, Division of Hematology, Department of Internal Medicine), Sharon L. Amacher, 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 mutations in zebrafish 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 transcripts 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. We have identified 169 genes that contain a proximal 3’UI in zebrafish, mouse and humans. Knockout of one such gene, foxo3b, in rbm8a and magoh mutants leads to partial phenotypic rescue. Finally, we show that β-globin reporters with 3’UTRs from proximal 3’UI-containing genes such as HNRNPD, FOXO3 and MEIS1 are upregulated in HeLa cells upon UPF1 knockdown. Overall, our findings define genes with 3’UTR introns within 50 nts of the stop codon as a new class of NMD targets, whose regulation by NMD maybe critical for their roles during development.
Keywords: mRNA decay, zebrafish development, motor neurons