Stop codon-proximal 3′UTR introns in vertebrates can elicit EJC-dependent Nonsense-Mediated mRNA Decay

The Exon Junction Complex (EJC) regulates many steps in post-transcriptional gene expression and is essential for cellular function and organismal development; however, EJC-regulated genes and genetic pathways during development remain largely unknown. To study EJC function during zebrafish development, we first established that zebrafish EJCs mainly bind [~]24 nucleotides upstream of exon-exon junctions, and are also detected at more distant non-canonical positions. We then generated mutations in two zebrafish EJC core genes, rbm8a and magoh, and observed that homozygous mutant embryos show paralysis, muscle disorganization, neural cell death, and motor neuron outgrowth defects. Coinciding with developmental defects, mRNAs subjected to Nonsense-Mediated mRNA Decay (NMD) due to translation termination [&ge;] 50 nts upstream of the last exon-exon junction are upregulated in EJC mutant embryos. Surprisingly, several transcripts containing 3'UTR introns (3'UI) < 50 nts downstream of a stop codon are also upregulated in EJC mutant embryos. These proximal 3'UI-containing transcripts are also upregulated in NMD-compromised zebrafish embryos, cultured human cells, and mouse embryonic stem cells. Loss of function of foxo3b, one of the upregulated proximal 3'UI-containing genes, partially rescues EJC mutant motor neuron outgrowth. In addition to foxo3b, 166 other genes contain a proximal 3'UI in zebrafish, mouse and humans, and these genes are enriched in nervous system development and RNA binding functions. A proximal 3'UI-containing 3'UTR from one of these genes, HNRNPD, is sufficient to reduce steady state transcript levels when fused to a {beta}-globin reporter in HeLa cells. Overall, our work shows that genes with stop codon-proximal 3'UIs encode a new class of EJC-regulated NMD targets with critical roles during vertebrate development.