In eukaryotes the majority of mRNAs have an m 7 G cap that is added cotranscriptionally and that plays an important role in many aspects of mRNA metabolism. The nuclear cap-binding complex (CBC; consisting of CBP20 and CBP80) mediates the stimulatory functions of the cap in pre-mRNA splicing, 3 end formation, and U snRNA export. As little is known about how nuclear CBC mediates the effects of the cap in higher eukaryotes, we have characterized proteins that interact with CBC in HeLa cell nuclear extracts as potential mediators of its function. Using cross-linking and coimmunoprecipitation, we show that eukaryotic translation initiation factor 4G (eIF4G), in addition to its function in the cytoplasm, is a nuclear CBC-interacting protein. We demonstrate that eIF4G interacts with CBC in vitro and that, in addition to its cytoplasmic localization, there is a significant nuclear pool of eIF4G in mammalian cells in vivo. Immunoprecipitation experiments suggest that, in contrast to the cytoplasmic pool, much of the nuclear eIF4G is not associated with eIF4E (translation cap binding protein of eIF4F) but is associated with CBC. While eIF4G stably associates with spliceosomes in vitro and shows close association with spliceosomal snRNPs and splicing factors in vivo, depletion studies show that it does not participate directly in the splicing reaction. Taken together the data indicate that nuclear eIF4G may be recruited to pre-mRNAs via its interaction with CBC and accompanies the mRNA to the cytoplasm, facilitating the switching of CBC for eIF4F. This may provide a mechanism to couple nuclear and cytoplasmic functions of the mRNA cap structure.RNAs transcribed by RNA polymerase II (pol II) are characterized by an inverted m 7 G(5Ј)ppp(5Ј)N cap. The capping of the pre-mRNA occurs cotranscriptionally (50, 52) and is achieved by recruitment of the capping enzyme to the phosphorylated C-terminal domain of the largest subunit of pol II (5,22,36). The cap contributes to many aspects of pol II transcript metabolism, including protection against 5Ј-3Ј exonucleases, facilitating efficient pre-mRNA splicing, 3Ј end formation, U snRNA and mRNA nuclear export, and translation of mRNAs (32,34).Two distinct families of cap binding proteins (CBPs) mediate the stimulatory effects of the cap structure. In the nucleus, the cap structure interacts with the nuclear cap-binding complex (CBC), a heterodimer consisting of two highly conserved polypeptides, CBP80 and CBP20 (20,23,54). CBC plays a direct role in pre-mRNA splicing, 3Ј end formation, and U snRNA export (reviewed in reference 32). In pre-mRNA splicing CBC promotes the association of U1 snRNP with the cap-proximal 5Ј splice site (31,33). In Saccharomyces cerevisiae, CBC interacts directly with yeast-specific components of the U1 snRNP (13,15). This contrasts with the mammalian system, where there is no evidence of a direct interaction between CBC and U1 snRNP (33). Although CBC is required for efficient 3Ј end cleavage of pre-mRNA, it is not required for polyadenylation per se (11)....

The initiation factor (eIF) 4E is regulated by modulating both the phosphorylation and the availability of the protein to participate in the initiation process. Here we show that either serum treatment or activation of the stress-activated protein kinase (JNK/SAPK) led to enhanced phosphorylation of eIF4E in quiescent NIH 3T3 cells. Although the immunosuppressant, rapamycin, was found to stabilize the association of eIF4E with its negative regulator, 4E-BP1, this drug did not prevent the early effects of serum stimulation on the overall rate of translation, polysome formation, the phosphorylation status of eIF4E, or the recruitment of eIF4E into the eIF4F complex. However, the rapid enhancement of eIF4E phosphorylation in response to serum was largely prevented by the inhibitor of mitogen-activated protein (MAP) kinase activation, PD98059. Activation of the JNK/SAPK signaling pathway with anisomycin resulted in enhanced phosphorylation of eIF4E, which was prevented by either rapamycin or the highly specific p38 MAP kinase inhibitor, SB203580. These data illustrate that multiple signaling pathways, including those of distinct members of the MAP kinase family, mediate the phosphorylation of eIF4E and that the association of eIF4E with 4E-BP1 does not necessarily prevent phosphorylation of eIF4E in vivo.

Initiation factor (eIF) 4G plays a key role in the regulation of translation, acting as a bridge between eIF4E and eIF3, to allow an mRNA molecule to associate with the 40S ribosomal subunit. In this study, we show that activation of the Fas/CD95 receptor complex in Jurkat cells induces the degradation of eIF4G, the inhibition of total protein synthesis and cell death. These responses were prevented by the caspase inhibitors, zVAD.FMK and zDEVD.FMK. We also show that, in contrast to Saccharomyces cerevisiae, although rapamycin caused a modest inhibition of protein synthesis it did not induce apoptosis or the cleavage of eIF4G. Studies with the specific inhibitor, SB203580, have shown that signalling through the p38 MAP kinase pathway is not required for either the Fas/CD95-induced cleavage of eIF4G or cell death. These data suggest that the cleavage of eIF4G and the inhibition of translation play an integral role in Fas/CD95-induced cell death in Jurkat cells.z 1998 Federation of European Biochemical Societies.

The question of whether translation initiation factor eIF4E and the complete eIF4G polypeptide are required for initiation dependent on the IRES (internal ribosome entry site) of hepatitis A virus (HAV) has been examined using in vitro translation in standard and eIF4G-depleted rabbit reticulocyte lysates. In agreement with previous publications, the HAV IRES is unique among all picornavirus IRESs in that it was inhibited if translation initiation factor eIF4G was cleaved by foot-and-mouth disease L-proteases. In addition, the HAV IRES was inhibited by addition of eIF4E-binding protein 1, which binds tightly to eIF4E and sequesters it, thus preventing its association with eIF4G. The HAV IRES was also inhibited by addition of m 7 GpppG cap analogue, irrespective of whether the RNA tested was capped or not. Thus, initiation on the HAV IRES requires that eIF4E be associated with eIF4G and that the cap-binding pocket of eIF4E be empty and unoccupied. This suggests two alternative models: (i) initiation requires a direct interaction between an internal site in the IRES and eIF4E/4G, an interaction which involves the cap-binding pocket of eIF4E in addition to any direct eIF4G-RNA interactions; or (ii) it requires eIF4G in a particular conformation which can be attained only if eIF4E is bound to it, with the cap-binding pocket of the eIF4E unoccupied.It is now generally accepted that picornavirus RNAs are translated by a mechanism of internal initiation, in which the ribosome enters directly at an internal site within the RNA rather than scanning from the physical 5Ј end (reviewed in reference 2). The 5Ј untranslated region (UTR) of the viral RNA has an IRES (internal ribosome entry site) about 450 nucleotides (nt) in length which is necessary and sufficient to promote internal ribosome entry and internal initiation. On the basis of primary and secondary structure conservation, the picornavirus IRESs can be divided into one minor and two major groups: (i) hepatitis A virus (HAV); (ii) entero-and rhinoviruses; and (iii) cardio-, aphtho-, and parechoviruses. Internal initiation of translation on the IRESs of the two major groups is thought to require all of the canonical initiation factors that are involved in the scanning mechanism except that eIF4E is completely redundant and the requirement for eIF4G can be fulfilled by just the C-terminal two-thirds fragment of this protein (26,27). Notably, the activity of these IRESs is not inhibited (and may even be actually stimulated in certain circumstances) when eIF4G is cleaved by entero-or rhinovirus 2A protease or foot-and-mouth disease virus (FMDV) L-protease (5, 6, 7, 28). These viral proteases cleave eIF4G to give (i) an N-terminal one-third fragment which has the site for interaction of eIF4G with eIF4E, the only translation initiation factor that binds directly to 5Ј caps, and also a site for binding poly(A)-binding protein; and (ii) a C-terminal two-thirds fragment which has two distinct sites for interaction with eIF4A, the RNA helicase initiation factor, and a site ...

Induction of apoptosis BJAB cells is accompanied by the rapid cleavage of protein synthesis eukaryotic initiation factor 4G and the appearance of a fragment of approximately 76 kDa. Inhibition of apoptotic proteases (caspases) has previously been shown to prevent the cleavage of eukaryotic initiation factor 4G. In MCF-7 breast carcinoma cells, which are deficient in caspase-3, eukaryotic initiation factor 4G is not cleaved but in vivo expression of caspase-3 restores eukaryotic initiation factor 4G cleavage following induction of apoptosis. Recombinant caspase-3 can also cleave eukaryotic initiation factor 4G to yield the 76 kDa fragment both in cell extracts and when the eukaryotic initiation factor 4G is presented in a purified eukaryotic initiation factor 4F complex. These results indicate that caspase-3 activity is necessary and sufficient for eukaryotic initiation factor 4G degradation.z 1999 Federation of European Biochemical Societies.