A novel RNA binding protein that interacts with NMDA R1 mRNA: regulation by ethanol.
Excitatory NMDA receptors are an important target of ethanol. Chronic ethanol exposure, in vivo and in vitro, increases polypeptide levels of NR1 subunit, the key subunit of functional NMDA receptors. In vitro, chronic ethanol treatment increases the half-life of NR1 mRNA and this observation is dependent on new protein synthesis. The present study was undertaken to locate cis-acting region(s) within the NR1 3'-untranslated region (UTR) and identify NR1 3'-UTR binding trans-acting proteins expressed in mouse fetal cortical neurons. Utilizing RNA gel shift assays we identified a 156-nt cis-acting region that binds to polysomal trans-acting proteins. This binding was highly specific as inclusion of cyclophilin RNA or tRNA did not interfere with cis-trans interactions. Importantly, the 3'-UTR binding activity was significantly up-regulated in the presence of ethanol. UV cross-link analysis detected three NR1 3'-UTR binding proteins and their molecular mass calculated by Northwestern analysis was approximately 88, 60 and 47 kDa, respectively. Northwestern analysis showed a significant up-regulation of the 88-kDa protein after chronic ethanol treatment. The 88-kDa protein was purified and identified by tandem mass spectrometry as the beta subunit of alpha glucosidase II (GIIbeta). That GIIbeta is indeed a trans-acting protein and binds specifically to 3'-UTR of NR1 mRNA was confirmed by RNA gel mobility supershift assays and immuno RT-PCR. Western blotting data established a significant increase of GIIbeta polypeptide in chronic ethanol-exposed fetal cortical neurons. We hypothesize that the identified cis-acting region and the associated RNA-binding proteins are important regulators of NR1 subunit gene expression.
Anji
Antje
A
Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA.
Kumari
Meena
M
D020413:3' Untranslated Regions / Q000187:drug effects; D000818:Animals; D015152:Blotting, Northern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D002540:Cerebral Cortex / Q000166:cytology; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004622:Embryo, Mammalian; D000431:Ethanol / Q000494:pharmacology*; D047468:Immunoprecipitation / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D011485:Protein Binding / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000502:physiology*; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000502:physiology*; D038621:Regulatory Sequences, Ribonucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014158:Transcription, Genetic
D020413:3' Untranslated Regions; D002492:Central Nervous System Depressants; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D016194:Receptors, N-Methyl-D-Aspartate; D038621:Regulatory Sequences, Ribonucleic Acid; D000431:Ethanol