title: Translational activity of N-methyl-D-aspartate receptor subunit NR1 mRNA in PC12 cells.
abstract: PC12 cells contain NR1 mRNA but lack significant expression of NR1 protein suggesting translational or posttranslational regulation. Translational activity of NR1 mRNA in PC12 cells was examined by sucrose gradient fractionation and by heterologous luciferase NR1 gene expression studies. The cosedimentation and association of NR1 mRNA with large polyribosomes (polysomes) confirmed the translatability of NR1 message in PC12 cells. Possible initiation and/or elongation defects during the translation of NR1 mRNAs were investigated by cyclohexamide treatment. The marked decline in the number of ribosomes associated with NR1 mRNA after prolonged exposure to cyclohexamide suggested that initiation was limiting translation of NR1 mRNA in PC12 cells. Consequently, the effect of the 5' and 3' untranslated regions (UTRs) on translation was examined using fusion constructs consisting of the luciferase coding region fused to either or both the 5' UTR and 3' UTR of NR1. The transfection of PC12 cells with the luciferase NR1-UTR fusion constructs revealed that the 3' UTR of NR1 had a significant inhibitory effect on luciferase expression. In contrast, the 5' UTR of NR1 had no inhibitory effect on mRNA translation in PC12 cells. The results from this study indicate that the translation of NR1 mRNA in PC12 cells may be impeded at initiation and this inhibition may be regulated at least in part through the 3' UTR of NR1.
authors.affiliation: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Awobuluyi
authors.forename: Rema
authors.initials: R
authors.lastname: Vazhappilly
authors.forename: Nikolaus J
authors.initials: NJ
authors.lastname: Sucher
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D015152:Blotting, Northern; D005591:Chemical Fractionation / Q000379:methods; D004622:Embryo, Mammalian; D006801:Humans; D007668:Kidney; D008156:Luciferases / Q000378:metabolism; D016716:PC12 Cells; D011132:Polyribosomes; D014176:Protein Biosynthesis / Q000502:physiology*; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013395:Sucrose; D014162:Transfection
chemical_list: D020121:5' Untranslated Regions; C409466:NR1 NMDA receptor; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; D013395:Sucrose; D008156:Luciferases