Nucleic Acids Research, 1995, Vol. 23, No. 12 2198-2205
© 1995
MOLECULAR BIOLOGY |
Autoregulation of poly(A)-binding protein synthesis in vitro
1Department of Biochemistry, University of Cambridge Tennis Court Road, Cambridge CB2 1QW, UK 2Dept. de Biologia Celular Universidade de Brasilia, Brasilia DF 70000, Brazil
* To whom correspondence should be addressed
Received February 22, 1995. Revised April 28, 1995. Accepted April 28, 1995.
The poly(A)-binding protein (PABP), in a complex with the 3' poly(A) tall of eukaryotic mRNAs, plays important roles In the control of translation and message stability. All known examples of PABP mRNAs contain an extensive A-rich sequence in their 5' untranslated regions. Studies in mammalian cells undergoing growth stimulation or terminal differentiation indicate that PABP expression is regulated at the translational level. Here we examine the hypothesis that synthesis of the PABP Is autogenously controlled. We show that the endogenous Inactive PABP mRNA in rabbit reticu-locytes can be specifically stimulated by addition of low concentrations of poly(A) and that this stimulation is also observed with In vitro transcribed human PABP mRNA. By deleting the A-rich region from the leader of human PABP mRNA and adding It upstream of the Initiator AUG in a reporter mRNA we show that the adenylate tract Is sufficient and necessary for mRNA repression and poly(A)-mediated activation in the reticulocyte cell-free system. UV cross-linking experiments demonstrate that the leader adenylate tract binds PABP. Furthermore, addition of recombinant GST-PABP to the cell-free system represses translation of mRNAs containing the A-rich sequence in their 5' UTR, but has no effect on control mRNA, We thus conclude that In vitro PABP binding to the A-rich sequence in the 5' UTR of PABP mRNA represses its own synthesis.
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