In many organisms, transcriptional and post-transcriptional regulation of components of pathways or processes has been reported. However, to date, there are few reports of translational co-regulation of multiple components of a developmental signaling pathway. Here, we show that an RNA element which we previously identified as a dorsal localization element (DLE) in the 3′UTR of zebrafish nodal-related1/squint (ndr1/sqt) ligand mRNA, is shared by the related ligand nodal-related2/cyclops (ndr2/cyc) and the nodal inhibitors, lefty1 (lft1) and lefty2 mRNAs. We investigated the activity of the DLEs through functional assays in live zebrafish embryos. The lft1 DLE localizes fluorescently labeled RNA similarly to the ndr1/sqt DLE. Similar to the ndr1/sqt 3′UTR, the lft1 and lft2 3′UTRs are bound by the RNA-binding protein (RBP) and translational repressor, Y-box binding protein 1 (Ybx1), whereas deletions in the DLE abolish binding to Ybx1. Analysis of zebrafish ybx1 mutants shows that Ybx1 represses lefty1 translation in embryos. CRISPR/Cas9-mediated inactivation of human YBX1 also results in human NODAL translational de-repression, suggesting broader conservation of the DLE RNA element/Ybx1 RBP module in regulation of Nodal signaling. Our findings demonstrate translational co-regulation of components of a signaling pathway by an RNA element conserved in both sequence and structure and an RBP, revealing a ‘translational regulon’.

Heart development is a complex process, starting from specification of cardiac precursors and formation of a linear tube to gradual progression to a functional beating organ. For normal heart development, many processes, including asymmetric positioning of the heart along the left-right (L/R) axis, cardiac growth, and cardiac valve morphogenesis must be completed successfully. Although heart development has been studied extensively, the mechanisms that control heart morphogenesis and valve formation are not fully understood. The pro-convertase FurinA is a key protein that functions in heart development in many vertebrates including zebrafish. How FurinA activity is regulated during heart development is not known. Through computational analysis of the zebrafish transcriptome, we identified a short sequence and structure RNA motif in a variant transcript of FurinA harbouring a long 3′ untranslated region (3′UTR). The alternative 3′UTR furina isoform is expressed at embryonic stages preceding organ positioning. Reporter localization and RNA-binding assays show that the furina 3′UTR forms complexes with the conserved RNA-binding protein and translational repressor Ybx1. Conditional mutant zebrafish embryos affecting ybx1 show premature and increased Furin reporter protein expression, abnormal cardiac morphogenesis and heart looping defects. Our mutant ybx1 hearts have an expanded atrioventricular canal, abnormal sino-atrial valves and many mutant embryos show retrograde blood flow from the ventricle to the atrium. This is similar to human heart valve regurgitation patients. Our findings show an essential function for the 3′UTR element/Ybx1 regulon in translational repression of FurinA, revealing a new upstream regulatory mechanism that controls embryonic heart development, and demonstrates the ybx1 mutant as a model to study cardiac valve development and function.