CNP2 MESSENGER-RNA DIRECTS SYNTHESIS OF BOTH CNP1 AND CNP2 POLYPEPTIDES

The ribosome scanning model for translational initiation predicts that eukaryotic mRNAs should, as a rule, be monocistronic. However, cases have recently been described of eukaryotic mRNAs producing more than o ne protein through alternative translational initiation at several dif ferent AUG codons. The present work reports the occurrence of two tran slational start sites on the mRNA encoding isoform 2 of the myelin mar ker enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in rat a nd mouse. We show that the CNP2 mRNA is able to direct synthesis of no t only CNP2, but also CNP1 polypeptide. Immunoprecipitation experiment s using a polyclonal antibody directed against CNP detect both CNP iso forms in tissues or cell lines expressing only the CNP2 transcript. Th us, the synthesis of CNP1 and CNP2 polypeptides must be encoded by the CNP2 transcript. In vitro translation of synthetic CNP2 mRNA demonstr ates that both CNP isoforms are synthesized by initiation at different AUG codons. Furthermore, by introducing mutations to ''switch off'' t ranslation from the second in-frame AUG codon in the CNP2 cDNA, and tr ansfecting 293T cells with those constructs, we are able to correlate the production of CNP1 and CNP2 with different translational start sit es. These results lead us to conclude that the CNP2 mRNA is able to pr oduce both CNP1 and CNP2 polypeptides. This investigation has altered our understanding of the temporal expression of the CNP protein isofor ms during development of the central nervous system (CNS). (C) 1997 Wi ley-Liss, Inc.