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 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.
Unité de Génétique Moléculaire des Virus Respiratoires, CNRS URA 1966, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France.
Y M
YM
Michel
D
D
Poncet
M
M
Piron
K M
KM
Kean
A M
AM
Borman
D000818:Animals; D001665:Binding Sites; D002457:Cell Extracts; D002474:Cell-Free System; D004686:Encephalomyocarditis virus / Q000235:genetics; D039603:Eukaryotic Initiation Factor-4G; D006367:HeLa Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011061:Poly A / Q000235:genetics / Q000378:metabolism*; D039102:Poly(A)-Binding Proteins; D011485:Protein Binding; D012315:RNA Caps / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes; D012270:Ribosomes / Q000378:metabolism; D012401:Rotavirus / Q000235:genetics / Q000502:physiology; D014461:Ultracentrifugation; D017361:Viral Nonstructural Proteins / Q000378:metabolism / Q000494:pharmacology
D002457:Cell Extracts; D039603:Eukaryotic Initiation Factor-4G; D010448:Peptide Initiation Factors; D039102:Poly(A)-Binding Proteins; D012315:RNA Caps; D016601:RNA-Binding Proteins; D017361:Viral Nonstructural Proteins; C089383:nsp3 protein, alphavirus; D011061:Poly A