Regulated translation of heparan sulfate N-acetylglucosamine N-deacetylase/N-sulfotransferase isozymes by structured 5′-untranslated regions and internal ribosome entry sites

We report the full-length 5'-untranslated region (5'-UTR) sequences of the four vertebrate heparan sulfate/heparin GlcNAc N-deacetylase/N-sulfotransferases (NDSTs) and their role in translational regulation in vivo and in vitro. All four NDST 5'-UTR sequences are unusually long, have a high degree of predicted secondary structure, and contain multiple upstream AUG codons, which together impose a major barrier to conventional, cap-dependent ribosomal scanning. At least two alternatively spliced forms of NDST2 differing in their 5'-UTRs exist, and two forms of NDST4 arise from alternative transcriptional start sites. The 5'-UTRs do not show any significant sequence similarity between isozymes, but possess highly conserved regions between mouse and human orthologs, pointing toward evolutionarily conserved functions. Expression of bicistronic vector constructs showed that the 5'-UTRs of NDST1-4 are capable of regulating translation differentially in vivo dependent on cell type and culture conditions. In vitro translation of a reporter gene located downstream of the UTRs demonstrated the presence of internal ribosome entry sites, providing an additional, cap-independent step in fine-tuning NDST expression. Comparative studies of NDST1-3 mRNAs and protein expression in brain and embryonic extracts revealed striking differences in translational efficiency. Other genes necessary for glycosaminoglycan synthesis in addition to the NDST isozymes have long, structured 5'-UTRs. Because several growth factors and morphogens that bind heparan sulfate also contain structured 5'-UTRs, translational regulation may coordinate the action of these factors and their heparan sulfate co-receptors.