Nicotinic stimulation modulates tyrosine hydroxylase mRNA half-life and protein binding to the 3'UTR in a manner that requires transcription.
Tyrosine hydroxylase (TH) expression increases in adrenal chromaffin cells treated with the nicotinic agonist, dimethylphenylpiperazinium (DMPP; 1 microM). We are using this response as a model of the changes in TH level that occur during increased cholinergic neural activity. Here we report a 4-fold increase in TH mRNA half-life in DMPP-treated cells chromaffin cells that is apparent when using a pulse-chase analysis to measure TH mRNA half-life. No increase is apparent using actinomycin D to measure half-life, indicating a requirement for ongoing transcription. Characterization of protein binding to the TH 3'UTR responsible for stabilization using labeled TH 3'UTR probes and electro-mobility shift assays shows the presence of two complexes both of which are increased by DMPP-treatment. The faster migrating complex (FMC) increases 2.5-fold and the slower migrating complex (SMC) increases 1.5-fold. Both changes are prevented by actinomycin D. Characterization of the protein binding to the TH UTR probes indicates SMC is disrupted by polyribonucleotides, poly (A) and poly (U), while binding to FMC is reduced by poly (CU). Separation of UV crosslinked RNA-protein complexes on SDS polyacrylamide gels shows FMC to contain a single protein whereas SMC contains three proteins. Northwesterns yielded similar results. Comparison of DMPP-induced protein binding with the poly C binding protein (PCBP) involved in hypoxia induced rat PC12 TH mRNA stability indicates none of the bovine UTR binding proteins are the PCBP. Thus, nicotinic stimulation produces a transcription-dependent increase in TH mRNA half-life that is mediated by previously unrecognized TH mRNA binding proteins.
Roe
David F
DF
Department of Neurobiology and Anatomy, The University of Texas Medical School, 5631 Fannin Street, Houston, TX 77030, USA. Jack.C.Waymire@uth.tmc.edu
Craviso
Gale L
GL
Waymire
Jack C
JC
D020413:3' Untranslated Regions / Q000378:metabolism*; D000313:Adrenal Medulla / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D015152:Blotting, Northern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects; D003609:Dactinomycin / Q000494:pharmacology; D004246:Dimethylphenylpiperazinium Iodide / Q000494:pharmacology*; D004347:Drug Interactions; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006207:Half-Life; D006801:Humans; D008969:Molecular Sequence Data; D018722:Nicotinic Agonists / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D009418:S100 Proteins / Q000378:metabolism; D013268:Stimulation, Chemical; D014158:Transcription, Genetic / Q000187:drug effects*; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism*
D020413:3' Untranslated Regions; D018722:Nicotinic Agonists; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D009418:S100 Proteins; D003609:Dactinomycin; D004246:Dimethylphenylpiperazinium Iodide; D014446:Tyrosine 3-Monooxygenase