Biochemical characterisation of cap-poly(A) synergy in rabbit reticulocyte lysates: the eIF4G-PABP interaction increases the functional affinity of eIF4E for the capped mRNA 5'-end.
The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to synergistically stimulate translation initiation in vivo. We recently described mammalian cytoplasmic extracts which, following ultracentrifugation to partially deplete them of ribosomes and associated initiation factors, reproduce cap-poly(A) synergy in vitro. Using these systems, we demonstrate that synergy requires interaction between the poly(A)-binding protein (PABP) and the eukaryotic initiation factor (eIF) 4F holoenzyme complex, which recognises the 5' cap. Here we further characterise the requirements and constraints of cap-poly(A) synergy in reticulocyte lysates by evaluating the effects of different parameters on synergy. The extent of extract depletion and the amounts of different initiation factors in depleted extracts were examined, as well as the effects of varying the concentrations of KCl, MgCl(2) and programming mRNA and of adding a cap analogue. The results presented demonstrate that maximal cap-poly(A) synergy requires: (i) limiting concentrations of ribosome-associated initiation factors; (ii) precise ratios of mRNA to translation machinery (low concentrations of ribosome-associated initiation factors and low, non-saturating mRNA concentrations); (iii) physiological concentrations of added KCl and MgCl(2). Additionally, we show that the eIF4G-PABP interaction on mRNAs which are capped and polyadenylated significantly increases the affinity of eIF4E for the 5' cap.
Borman
A M
AM
Unité de Génétique Moléculaire des Virus Respiratoires, CNRS URA 1966, Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France.
Michel
Y M
YM
Kean
K M
KM
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002457:Cell Extracts; D002474:Cell-Free System; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D020035:Holoenzymes / Q000378:metabolism; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000378:metabolism*; D011061:Poly A / Q000235:genetics / Q000378:metabolism*; D039102:Poly(A)-Binding Proteins; D011189:Potassium Chloride / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding / Q000187:drug effects; D012315:RNA Caps / Q000235:genetics / Q000378:metabolism*; D020871:RNA Stability; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D013816:Thermodynamics
D002457:Cell Extracts; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D020035:Holoenzymes; D010448:Peptide Initiation Factors; D039102:Poly(A)-Binding Proteins; D012315:RNA Caps; D016601:RNA-Binding Proteins; D015636:Magnesium Chloride; D011061:Poly A; D011189:Potassium Chloride