Translational Control of Poly(A)-Binding Protein Expression

Authors


  • Note. Preliminary results on the autoregulation of poly(A)-binding protein mRNA translation were first presented by these authors at the Cold Spring Harbour Laboratory Translational Control Meeting on September 9–13, 1992.

J. Bag, Department of Molecular Biology and Genetics, University of Guelph, Ontario, Canada N1G 2W1

Abstract

Poly(A)-binding protein (PABP) is important for translation of eukaryotic mRNA and may be involved in shortening of its poly(A) tract. In many eukaryotic cells, this mRNA is inefficiently translated. The 5′ untranslated region (UTR) of PABP mRNA has several adenine-rich regions which may serve as the PABP-binding sites to control its translation by a feed-back mechanism. This postulate was tested by using in vitro transcribed PABP mRNA and a rabbit reticulocyte lysate cell-free system. Results of our studies show that removal of the putative PABP-binding sites from the 5′ UTR of this mRNA enhances its translation in the rabbit reticulocyte cell-free system. Furthermore, in vitro translation of the full-length PABP mRNA was inhibited by addition of purified PABP to the cell-free system. In contrast, translation of truncated mRNA lacking the putative PABP-binding sites at the 5′ UTR was not inhibited by exogenous PABP. We have also tested the ability of purified PABP to bind to the 5′ UTR of PABP mRNA using ultraviolet-mediated covalent cross-linking of RNA and proteins in vitro. Our results show that exogenous PABP binds to the 5′ UTR of its full-length mRNA. Furthermore, incubation of PABP mRNA in rabbit reticulocyte lysate also led to binding of the endogenous PABP within the first 223 nucleotides of the 5′ UTR. The adenine-rich regions are located within this segment of PABP mRNA. Following incubation of PABP mRNA in the reticulocyte lysate cell-free system under conditions of mRNA translation, the polysomal and non-translated free mRNA fractions were separated by centrifugation. Analysis of free and polysomal mRNA-protein (mRNP) complexes following ultraviolet-induced cross-linking showed that the free mRNP population was preferentially enriched in PABP. Results of our studies, therefore, suggest that PABP mRNA translation may be repressed by a unique feed-back mechanism.

Abbreviations
PABP

poly(A)-binding protein

UTR

untranslated region

mRNP

mRNA-protein complex

eIF

eukaryotic initiation factor

GTS

glutathione S-transferase

IPTG

isopropyl-β-d-thiogalactopyranoside