An Efficient System for Cap- and Poly(A)-Dependent Translation In Vitro
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The 3′ poly(A) tail of eukaryotic messenger RNAs (mRNAs) acts synergistically with the 5′ cap structure to enhance translation.
This phenomenon has been explained by the simultaneous binding of poly(A)-binding protein (PABP) and a cap-binding protein
(eIF4E) to eIF4G that results in the circularization of the mRNA (closed-loop model). We developed a robust cell-free protein
synthesis system to study poly(A)-dependent translation. In nuclease-treated extracts of Krebs-2 ascites cells, the mRNA poly(A)
tail and the cap structure synergistically stimulate translation. We also describe an efficient procedure for depleting PABP
from translation extracts. Greater than 98% of PABP can be depleted from extracts by preincubation with either of the PABP-interacting
proteins (Paip2 or Paip1) coupled to beads, and these depleted extracts fail to support efficient translation of poly(A)+
mRNAs. Translation activity is restored to depleted extracts by the addition of recombinant PABP.