An Efficient System for Cap- and Poly(A)-Dependent Translation In Vitro

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.