No abstract

Eukaryotic initiation factor (elF) 4A functions as a subunit of the initiation factor complex elF4F, which mediates the binding of mRNA to the ribosome. 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 thought that the function of elF4A during translation initiation is to unwind the mRNA secondary structure in the 5' UTR to facilitate ribosome binding. However, the evidence to support this hypothesis is rather indirect, and it was reported that elF4A is also required for the translation of mRNAs possessing minimal 5' UTR secondary structure. Were this hypothesis correct, the requirement for elF4A should correlate with the degree of mRNA secondary structure. To test this hypothesis, the effect of a dominant-negative mutant of mammalian elF4A on translation of mRNAs with various degrees of secondary structure was studied in vitro. Here, we show that mRNAs containing stable secondary structure in the 5' untranslated region are more susceptible to inhibition by the elF4A mutant. The mutant protein also strongly inhibits translation from several picornavirus internal ribosome entry sites (IRES), although to different extents. UV crosslinking of elF4F subunits and elF4B to the mRNA cap structure is dramatically reduced by the elF4A mutant and RNA secondary structure. Finally, the elF4A mutant forms a more stable complex with elF4G, as compared to the wild-type elF4A, thus explaining the mechanism by which substoichiometric amounts of mutant elF4A inhibit translation.

In the initiation of translation in eukaryotes, binding of the small ribosomal subunit to the messenger RNA results from recognition of the 5' cap structure (m7GpppX) of the mRNA by the cap-binding complex eIF4F. eIF4F is itself a three-subunit complex comprising the cap-binding protein eIF4E, eIF4A, an ATP-dependent RNA helicase, and eIF4G, which interacts with both eIF4A and eIF4E and enhances cap binding by eIF4E. The mRNA 3' polyadenylate tail and the associated poly(A)-binding protein (PABP) also regulate translational initiation, probably by interacting with the 5' end of the mRNA. In yeast and plants, PABP interacts with eIF4G but no such interaction has been reported in mammalian cells. Here, we describe a new human PABP-interacting protein, PAIP-I, whose sequence is similar to the central portion of eIF4G and which interacts with eIF4A. Overexpression of PAIP-1 in COS-7 cells stimulates translation, perhaps by providing a physical link between the mRNA termini.

No abstract

The cap structure, m 7 GpppN, is present at the 5-end of all eukaryotic cellular (except organellar) mRNAs. Initiation of translation is mediated by the multisubunit initiation factor eIF4F, which binds the cap structure via its eIF4E subunit and facilitates the binding of mRNA to ribosomes. Here, we used recombinant proteins to reconstitute the cap recognition activity of eIF4F in vitro. We demonstrate that the interaction of eIF4E with the mRNA 5-cap structure is dramatically enhanced by eIF4G, as determined by a UV-induced cross-linking assay. Furthermore, assembly of the eIF4F complex at the cap structure, as well as ATP hydrolysis, is shown to be a requisite for the cross-linking of another initiation factor, eIF4B, to the cap structure. In addition, the stimulatory effect of eIF4G on the cap recognition of eIF4E is inhibited by the translational repressor, 4E-BP1. These results suggest that eIF4E initially interacts with the mRNA cap structure as part of the eIF4F complex.Cap-dependent binding of ribosomes to mRNA is mediated by several initiation factors, eIF4F, eIF4A, and eIF4B, and requires energy derived from ATP hydrolysis (1). eIF4F is a three-subunit complex composed of (i) eIF4E, (ii) eIF4A, and (iii) eIF4G. eIF4E is a 24-kDa polypeptide that specifically interacts with the 5Ј-cap structure (m 7 GpppN; where N is any nucleotide) (2). eIF4A is a 50-kDa protein that exhibits RNAdependent ATPase activity and, in conjunction with eIF4B, RNA helicase activity (3, 4). eIF4G is a 154-kDa polypeptide that binds to both eIF4E and eIF4A (5, 6). eIF4G also exhibits sequence-nonspecific RNA binding activity that is most probably responsible for the RNA binding activity of eIF4F (7) 1 . eIF4E activity is regulated by two proteins, termed 4E-BP1 and 4E-BP2 (8, 9). Interaction of 4E-BP1 with eIF4E inhibits specifically cap-dependent translation (9). 4E-BPs are rapidly hyperphosphorylated in cells following treatment with insulin and growth factors (10, 11). The phosphorylation of 4E-BPs decreases the association of 4E-BP1 with eIF4E (9). Consequently, phosphorylation of 4E-BPs leads to stimulation of translation. 4E-BP1 competes with eIF4G for binding to eIF4E through similar sequence motifs (12). Furthermore, the association of 4E-BP1 with eIF4E prevents the in vitro phosphorylation of eIF4E by protein kinase C, raising the possibility of a temporal relationship between eIF4E binding to 4E-BPs and eIF4E phosphorylation (13).Two models were proposed for the pathway of eIF4F assembly and subsequent ribosome binding. One model posits that the first step of ribosome binding is the interaction between eIF4F and the mRNA cap structure (1). According to this model, eIF4F in combination with eIF4B and eIF4A, unwinds secondary structure in the 5Ј-untranslated region of the mRNA, to create a single-stranded region of RNA, which serves as a binding site for the 43 S preinitiation complex. eIF4B and eIF4A were shown to cross-link to the cap structure only in the presence of eIF4F in a process that requires ATP hydrolysis (1...