The requirement for eukaryotic initiation factor 4A (eIF4A) in translationis in direct proportion to the degree of mRNA 5 ' secondary structure

Eukaryotic initiation factor (elF) 4A functions as a subunit of the initiat ion factor complex elF4F, which mediates the binding of mRNA to the ribosom e, elF4A possesses ATPase and RNA helicase activities and is the prototype for a large family of putative RNA helicases (the DEAD box family). It is t hought that the function of elF4A during translation initiation is to unwin d the mRNA secondary structure in the 5 ' UTR to facilitate ribosome bindin g. However, the evidence to support this hypothesis is rather indirect, and it was reported that elF4A is also required for the translation of mRNAs p ossessing minimal 5 ' UTR secondary structure. Were this hypothesis correct , the requirement for elF4A should correlate with the degree of mRNA second ary structure. To test this hypothesis, the effect of a dominant-negative m utant of mammalian elF4A on translation of mRNAs with various degrees of se condary structure was studied in vitro. Here, we show that mRNAs containing stable secondary structure in the 5 ' untranslated region are more suscept ible to inhibition by the elF4A mutant. The mutant protein also strongly in hibits translation from several picornavirus internal ribosome entry sites (IRES), although to different extents. UV crosslinking of elF4F subunits an d elF4B to the mRNA cap structure is dramatically reduced by the elF4A muta nt and RNA secondary structure. Finally, the elF4A mutant forms a more stab le complex with elF4G, as compared to the wild-type elF4A, thus explaining the mechanism by which substoichiometric amounts of mutant elF4A inhibit tr anslation.