Dominant negative mutants of mammalian translation initiation factor eIF-4A define a critical role for eIF-4F in cap-dependent and cap-independent initiation of translation.

Abstract

Eukaryotic translation initiation factor‐4A (eIF‐4A) plays a critical role in binding of eukaryotic mRNAs to ribosomes. It has been biochemically characterized as an RNA‐dependent ATPase and RNA helicase and is a prototype for a growing family of putative RNA helicases termed the DEAD box family. It is required for mRNA‐ribosome binding both in its free form and as a subunit of the cap binding protein complex, eIF‐4F. To gain further understanding into the mechanism of action of eIF‐4A in mRNA‐ribosome binding, defective eIF‐4A mutants were tested for their abilities to function in a dominant negative manner in a rabbit reticulocyte translation system. Several mutants were demonstrated to be potent inhibitors of translation. Addition of mutant eIF‐4A to a rabbit reticulocyte translation system strongly inhibited translation of all mRNAs studied including those translated by a cap‐independent internal initiation mechanism. Addition of eIF‐4A or eIF‐4F relieved inhibition of translation, but eIF‐4F was six times more effective than eIF‐4A, whereas eIF‐4B or other translation factors failed to relieve the inhibition. Kinetic experiments demonstrated that mutant eIF‐4A is defective in recycling through eIF‐4F, thus explaining the dramatic inhibition of translation. Mutant eIF‐4A proteins also inhibited eIF‐4F‐dependent, but not eIF‐4A‐dependent RNA helicase activity. Taken together these results suggest that eIF‐4A functions primarily as a subunit of eIF‐4F, and that singular eIF‐4A is required to recycle through the complex during translation. Surprisingly, eIF‐4F, which binds to the cap structure, appears to be also required for the translation of naturally uncapped mRNAs.

A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initation of hepatitis C and classical swine fever virus RNAs

“…Both processes are inhibited by the transdominant eIF4A (R362Q) mutant; inhibition is relieved by eIF4F, and less effectively, by eIF4A (Pause et al 1994). The data above indicate that 43S ribosomal complexes bind to HCV and CSFV IRESs without eIF4A, eIF4B, or eIF4F and can then bind 60S ribosomal subunits to form active 80S complexes.…”

“…The processes of ribosomal binding to these IRESs, positioning of the ribosome at the initiation codon, and formation of active 80S complexes did not require the initiation factors eIF4A, eIF4B, and eIF4F and were not influenced by them. Initiation mediated by these two IRESs therefore differs fundamentally from initiation on all other eukaryotic mRNAs, including even internal initiation mediated by the EMCV IRES, which depends absolutely on one or more of these factors (Trachsel et al 1977;Blum et al 1989;Pause et al 1994;Pestova et al 1996a,b). This unexpected observation was supported strongly by the lack of effect of the eIF4A R362Q mutant on HCV and CSFV translation in RRL.…”

“…This unexpected observation was supported strongly by the lack of effect of the eIF4A R362Q mutant on HCV and CSFV translation in RRL. This eIF4A mutant is a trans-dominant inhibitor of capmediated and EMCV IRES-mediated initiation (Pause et al 1994). eIF4A, eIF4B, and eIF4F are involved in ATPdependent attachment of 40S subunits to conventional eukaryotic mRNAs and in ATP-dependent ribosomal scanning from the binding site to the initiation codon (Merrick 1992).…”

The role of c-myc in cellular growth control

“…Intriguingly, the DEAD box helicases comprise a family of proteins that catalyze unwinding of RNA. Members of this family include proteins involved in both translation initiation (eIF4A) and ribosomal RNA processing (Pause et al, 1994;Rozen et al, 1990). Although the biochemical function of this Myc target is currently unknown, its homologue in Drosophila has been named pitchoune as a consequence of the small size phenotype which results from its loss (Za ran et al, 1998).…”

Starting the protein synthesis machine: eukaryotic translation initiation

“…An important feature of eIF4A's function is also an apparent need to exchange eIF4F-bound with free factor for efficient unwinding. (32,33) eIF4B is a homodimer that can bind RNA by virtue of an N-terminal RNA recognition motif (RRM) and a C-terminal arginine-rich motif. (12) Another recently identified factor, eIF4H, has homology to the RRM domain of eIF4B and has also been shown to stimulate ATPase and helicase activity of eIF4A.…”