Identification of a Nucleic Acid Binding Domain in Eukaryotic Initiation Factor eIFiso4G from Wheat

Abstract

Higher plants have two complexes that bind the m 7 Gcap structure of mRNA and mediate interactions between mRNA and ribosomal subunits, designated eIF4F and eIFiso4F. Both complexes contain a small subunit that binds the 5-cap structure of mRNA, and a large subunit, eIF4G or eIFiso4G, that binds other translation factors and RNA. Sequence-specific proteases were used to cleave native cap-binding complexes into structural domains, which were purified by affinity chromatography. We show here that eIFiso4G contains a central pro- The group 4 translation initiation factors, eIF4A, 1 B, E, and G, bind and recruit mRNA to 43 S pre-initiation complexes for translation (reviewed in Refs. 1 and 2). Association of eIF4A and eIF4B confers ATPase, RNA helicase, and RNA annealing activities on the intact complex (1-3) and is thought to be necessary for melting of RNA secondary structure and ribosome scanning to the AUG start codon. eIF4E binds to the 5Ј-cap structure of mRNAs and associates tightly with eIF4G. These two factors together form the core of the mRNA-binding complex, designated eIF4F. Higher plants have two such complexes: eIF4F, which is homologous to the mammalian complex, and eIFiso4F, which is ϳ10-fold more abundant (4, 5). eIF4G and eIFiso4G differ ϳ2-fold in molecular mass and show only 25-30% sequence similarity overall, with ϳ50% similarity in six small homologous regions (6). Yet, despite this substantial divergence, these two factors have very similar functions (1). Either factor alone is sufficient for cap-dependent initiation in vitro (7,8). eIFiso4G and eIFiso4E have been cloned and sequenced from wheat and have deduced molecular masses of 86.5 and 23.5 kDa, respectively (6).The eIF4G and eIFiso4G subunits of the mRNA-binding complexes act as a central scaffold for binding of other translation factors, proteins, co-factors, and RNAs. These include binding sites within mammalian eIF4G for eIF4E (aa 409 -457) (9), eIF3 (aa 480 -886) (10), and two sites for eIF4A (aa 887-1402 and 478 -883) (10, 11). Plant eIFiso4G also binds eIFiso4E near its N terminus (aa 52-90) (12) in a small region with sequence homology to the mammalian factor. Binding of eIF4G and eIFiso4G to RNAs, distinct from the cap recognition function of the intact complex, has been demonstrated by several laboratories and appears to play a critical role in the cap-independent initiation of viral RNAs. Sha et al. (13) calculated the equilibrium binding constants of recombinant wheat eIFiso4G for both capped and uncapped mRNAs. They found that the binding constants for uncapped message were comparable to those of eIFiso4E and eIFiso4F for capped mRNA, clearly indicating that the large subunit also binds mRNA efficiently and that it does not discriminate between capped and uncapped messages. These findings are consistent with the role of eIF4G in the cap-independent translation of viral mRNAs, following cleavage and loss of the eIF4E binding domain by viral proteases (10, 14). In functional assays for initiation complex formation, Pestova et...

The 3′ cap-independent translation element of Barley yellow dwarf virus binds eIF4F via the eIF4G subunit to initiate translation

“…Thus, we deduce that the C-terminal half of eIF4G contains the BTE binding site(s). This is consistent with the positions of the functional domains identified in wheat eIF4G (Kim et al 1999;Gallie and Browning 2001), and the well-characterized mammalian eIF4G (Prevot et al 2003a;Hinton et al 2007;Pestova et al 2007). Most of the key domains required for eIF4G function, including domains for binding RNA, eIF4A, and eIF3 are located in the C-terminal half.…”

Interaction of the eIF4G initiation factor with the aphthovirus IRES is essential for internal translation initiation in vivo

“…The interaction between FMDV IRES and eIF4G also occurred efficiently with the Lb-processed form of this initiation factor as it is present in infected cells+ This result demonstrates that this RNA-protein interaction was supported by the C-terminal fragment of the proteolytically processed protein (Fig+ 2), in agreement with data reported for the EMCV IRES (Kolupaeva et al+, 1998)+ The increased intensity of the crosslinked polypeptide in the Lb-processed extract compared to the intact eIF4G is consistent with the stimulation of internal initiation efficiency by the C-terminal fragment of eIF4G-IRES interaction determines internal translation initiationeIF4G shown for several IRES elements (Ohlmann et al+, 1996;Borman et al+, 1997)+ Binding of eIF4G to the FMDV IRES is independent of eIF4B interaction, as demonstrated with probe 4, which interacted with eIF4G alone (Fig+ 4)+ Furthermore, a similar intensity of the UV-crosslinked polypeptide was observed with probes 4 and 4-5, indicating that efficient binding of eIF4B does not prevent eIF4G-IRES interaction+ Taken together, these results suggest that eIF4B and eIF4G may interact with the IRES independently of each other, recognizing different residues within an overlapping region of the 39 end of the IRES+ Both eIF4B and eIF4G proteins contain RRM (Goyer et al+, 1993;Naranda et al+, 1994;Méthot et al+, 1994)+ However, they behave differently in their interaction with RNA (Méthot et al+, 1994;Naranda et al+, 1994;Kim et al+, 1999) and the specific sequences recognized in the RNA-binding sites are not well characterized yet+ Regarding the eIF4B binding sequences in the RNA, in vitro selection experiments carried out to isolate high affinity RNA ligands led to the identification of several molecules, all containing hairpins with internal loops (Méthot et al+, 1996a)+ Within the IRES of FMDV we have found that domain 5 is absolutely required for binding to eIF4B, as the transcript containing only domain 4 did not crosslink to eIF4B+ Interestingly, the secondary structure of this short domain (Fig+ 5A) resembles that of the high affinity ligands described previously (Méthot et al+, 1996a)+ The RRM of eIF4G lies within the central region of the protein, which is conserved among all the eIF4G-related molecules (Gingras et al+, 1999), indicating that RNA recognition is one of the essential features of these proteins+ It is noteworthy that the eIF4G C-terminal fragment contains the RRM (Fig+ 2), in agreement with the suggestion that IRES-eIF4G interaction may involve the RRM+ At present, the sequences in the RNA that constitute the binding site of eIF4G are poorly known+ RNA sequences in domain J-K of the EMCV IRES (equivalent to the FMDV domain 4) including the oligo(A) bulge were protected from dimethyl-sulfate modification after interaction with eIF4G (Kolupaeva et al+, 1998)+ In agreement with our finding that the primary sequence of the FMDV internal AA loop is essential for eIF4G interaction, the corresponding sequences in the EMCV IRES were also protected from dimethyl-sulfate modification+ Thus, the conservation of the eIF4G RNA recognition sites in FMDV and EMCV I...…”

RNA-binding activity of translation initiation factor eIF4G1 from Saccharomyces cerevisiae

“…Indeed, a nucleic-acid-binding domain was found in wheat eIFiso4G (Kim et al 1999). Furthermore, putative RNA-binding motifs were identified in yeast eIF4G by amino acid sequence inspection (Goyer et al 1993).…”

“…We verified the RNA-binding activity of eIF4G1 and eIF4G1 fragments by a different method and chose adsorption to poly(U)-agarose (Kim et al 1999), which has the advantage over the filter-binding assay of allowing visualization of proteins bound to RNA. We tested full-length eIF4G1 1-952 and the fragments eIF4G1 1-82 , eIF4G1 , and eIF4G1 883-952 carrying RNA-binding sites as determined by filter-binding assays.…”