Cap-Poly(A) synergy in mammalian cell-free extracts. Investigation of the requirements for poly(A)-mediated stimulation of translation initiation

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to stimulate synergistically translation initiation in vivo, a phenomenon observed to date in vitro only in translation systems containing endogenous competitor mRNAs. Here we describe nuclease-treated rabbit reticulocyte lysates...

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Bibliographic Details
Published in:The Journal of biological chemistry 2000-10, Vol.275 (41), p.32268-32276
Main Authors: Michel, Y M, Poncet, D, Piron, M, Kean, K M, Borman, A M
Format: Article
Language:English
Subjects:
Animals
Binding Sites
Biochemistry
Biochemistry, Molecular Biology
Cell Extracts
Cell-Free System
Encephalomyocarditis virus
Encephalomyocarditis virus - genetics
Eukaryotic Initiation Factor-4G
HeLa Cells
Humans
initiation factor eIF-4G
Life Sciences
Nucleic Acid Conformation
PABP protein
Peptide Chain Initiation, Translational
Peptide Initiation Factors
Peptide Initiation Factors - metabolism
Plasmids
Plasmids - genetics
Poly A
Poly A - genetics
Poly A - metabolism
Poly(A)-Binding Proteins
Protein Binding
Rabbits
Reticulocytes
Ribosomes
Ribosomes - metabolism
RNA Caps
RNA Caps - genetics
RNA Caps - metabolism
RNA-Binding Proteins
RNA-Binding Proteins - metabolism
Rotavirus
Rotavirus - genetics
Rotavirus - physiology
Ultracentrifugation
Viral Nonstructural Proteins
Viral Nonstructural Proteins - metabolism
Viral Nonstructural Proteins - pharmacology
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Summary:The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to stimulate synergistically translation initiation in vivo, a phenomenon observed to date in vitro only in translation systems containing endogenous competitor mRNAs. Here we describe nuclease-treated rabbit reticulocyte lysates and HeLa cell cytoplasmic extracts that reproduce cap-poly(A) synergy in the absence of such competitor RNAs. Extracts were rendered poly(A)-dependent by ultracentrifugation to partially deplete them of ribosomes and associated initiation factors. Under optimal conditions, values for synergy in reticulocyte lysates approached 10-fold. By using this system, we investigated the molecular mechanism of poly(A) stimulation of translation. Maximal cap-poly(A) cooperativity required the integrity of the eukaryotic initiation factor 4G-poly(A)-binding protein (eIF4G-PABP) interaction, suggesting that synergy results from mRNA circularization. In addition, polyadenylation stimulated uncapped cellular mRNA translation and that driven by the encephalomyocarditis virus internal ribosome entry segment (IRES). These effects of poly(A) were also sensitive to disruption of the eIF4G-PABP interaction, suggesting that 5'-3' end cross-talk is functionally conserved between classical mRNAs and an IRES-containing mRNA. Finally, we demonstrate that a rotaviral non-structural protein that evicts PABP from eIF4G is capable of provoking the shut-off of host cell translation seen during rotavirus infection.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M004304200