CNP2 mRNA 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 one protein through alternative translational initiation at several different AUG codons. The present work reports the occurrence of two translational start sites on the mRNA encoding isoform 2 of the myelin marker enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in rat and mouse. We show that the CNP2 mRNA is able to direct synthesis of not only CNP2, but also CNP1 polypeptide. Immunoprecipitation experiments using a polyclonal antibody directed against CNP detect both CNP isoforms in tissues or cell lines expressing only the CNP2 transcript. Thus, the synthesis of CNP1 and CNP2 polypeptides must be encoded by the CNP2 transcript. In vitro translation of synthetic CNP2 mRNA demonstrates that both CNP isoforms are synthesized by initiation at different AUG codons. Furthermore, by introducing mutations to "switch off" translation from the second in-frame AUG codon in the CNP2 cDNA, and transfecting 293T cells with those constructs, we are able to correlate the production of CNP1 and CNP2 with different translational start sites. These results lead us to conclude that the CNP2 mRNA is able to produce both CNP1 and CNP2 polypeptides. This investigation has altered our understanding of the temporal expression of the CNP protein isoforms during development of the central nervous system (CNS).
O'Neill
R C
RC
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Minuk
J
J
Cox
M E
ME
Braun
P E
PE
Gravel
M
M
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D007527:Isoenzymes / Q000235:genetics*; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010442:Peptide Chain Initiation, Translational / Q000235:genetics; D010446:Peptide Fragments / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000502:physiology*; D051381:Rats
D007527:Isoenzymes; D010446:Peptide Fragments; D012333:RNA, Messenger; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases