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
AUTOR(ES)
Borman, Andrew M.
FONTE
Oxford University Press
RESUMO
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, MgCl2 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 MgCl2. 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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=113128Documentos Relacionados
- Truncated initiation factor eIF4G lacking an eIF4E binding site can support capped mRNA translation
- Purifying mRNAs with a high-affinity eIF4E mutant identifies the short 3′ poly(A) end phenotype
- PML RING suppresses oncogenic transformation by reducing the affinity of eIF4E for mRNA
- Structure of a Viral Cap-independent Translation Element That Functions via High Affinity Binding to the eIF4E Subunit of eIF4F*S⃞
- A novel shuttling protein, 4E-T, mediates the nuclear import of the mRNA 5′ cap-binding protein, eIF4E