Identification of two proteins that bind to a pyrimidine-rich sequence in the 3'-untranslated region of GAP-43 mRNA.
GAP-43 is a membrane phosphoprotein that is important for the development and plasticity of neural connections. In undifferentiated PC12 pheochromocytoma cells, GAP-43 mRNA degrades rapidly ( t = 5 h), but becomes stable when cells are treated with nerve growth factor. To identify trans- acting factors that may influence mRNA stability, we combined column chromatography and gel mobility shift assays to isolate GAP-43 mRNA binding proteins from neonatal bovine brain tissue. This resulted in the isolation of two proteins that bind specifically and competitively to a pyrimidine-rich sequence in the 3'-untranslated region of GAP-43 mRNA. Partial amino acid sequencing revealed that one of the RNA binding proteins coincides with FBP (far upstream element binding protein), previously characterized as a protein that resembles hnRNP K and which binds to a single-stranded, pyrimidine-rich DNA sequence upstream of the c -myc gene to activate its expression. The other binding protein shares sequence homology with PTB, a polypyrimidine tract binding protein implicated in RNA splicing and regulation of translation initiation. The two proteins bind to a 26 nt pyrimidine-rich sequence lying 300 nt downstream of the end of the coding region, in an area shown by others to confer instability on a reporter mRNA in transient transfection assays. We therefore propose that FBP and the PTB-like protein may compete for binding at the same site to influence the stability of GAP-43 mRNA.
Irwin
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Department of Neurosurgery, Children's Hospital, Boston, MA 02115, USA. irwin_c@a1.tch.harvard.edu
Baekelandt
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Goritchenko
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Benowitz
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LI
D000310:Adrenal Gland Neoplasms; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D003198:Computer Simulation; D019922:GAP-43 Protein; D016259:Genes, myc; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D016716:PC12 Cells; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010673:Pheochromocytoma; D010750:Phosphoproteins / Q000096:biosynthesis; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D012689:Sequence Homology, Nucleic Acid
D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins