The major mRNA-associated protein YB-1 is a potent 5' cap-dependent mRNA stabilizer.

mRNA silencing and storage play an important role in gene expression under diverse circumstances, such as throughout early metazoan development and in response to many types of environmental stress. Here we demonstrate that the major mRNA-associated protein YB-1, also termed p50, is a potent cap-dependent mRNA stabilizer. YB-1 addition or overexpression dramatically increases mRNA stability in vitro and in vivo, whereas YB-1 depletion results in accelerated mRNA decay. The cold shock domain of YB-1 is responsible for the mRNA stabilizing activity, and a blocked mRNA 5' end is required for YB-1-mediated stabilization. Significantly, exogenously added YB-1 destabilizes the interaction of the cap binding protein, eIF4E, with the mRNA cap structure. Conversely, sequestration of eIF4E from the cap increases the association of endogenous YB-1 with mRNA at or near the cap, and significantly enhances mRNA stability. These data support a model whereby down-regulation of eIF4E activity or increasing the YB-1 mRNA binding activity or concentration in cells activates a general default pathway for mRNA stabilization.

Translational silencing of ceruloplasmin requires the essential elements of mRNA circularization: poly(A) tail, poly(A)-binding protein, and eukaryotic translation initiation factor 4G.

Ceruloplasmin (Cp) is a glycoprotein secreted by the liver and monocytic cells and probably plays roles in inflammation and iron metabolism. We showed previously that gamma interferon (IFN-gamma) induced Cp synthesis by human U937 monocytic cells but that the synthesis was subsequently halted by a transcript-specific translational silencing mechanism involving the binding of a cytosolic factor(s) to the Cp mRNA 3' untranslated region (UTR). To investigate how protein interactions at the Cp 3'-UTR inhibit translation initiation at the distant 5' end, we considered the "closed-loop" model of mRNA translation. In this model, the transcript termini are brought together by interactions of poly(A)-binding protein (PABP) with both the poly(A) tail and initiation factor eIF4G. The effect of these elements on Cp translational control was tested using chimeric reporter transcripts in rabbit reticulocyte lysates. The requirement for poly(A) was shown since the cytosolic inhibitor from IFN-gamma-treated cells minimally inhibited the translation of a luciferase reporter upstream of the Cp 3'-UTR but almost completely blocked the translation of a transcript containing a poly(A) tail. Likewise, a requirement for poly(A) was shown for silencing of endogenous Cp mRNA. We considered the possibility that the cytosolic inhibitor blocked the interaction of PABP with the poly(A) tail or with eIF4G. We found that neither of these interactions were inhibited, as shown by immunoprecipitation of PABP followed by quantitation of the poly(A) tail by reverse transcription-PCR and of eIF4G by immunoblot analysis. We considered the alternate possibility that these interactions were required for translational silencing. When PABP was depleted from the reticulocyte lysate with anti-human PABP antibody, the cytosolic factor did not inhibit translation of the chimeric reporter, thus showing the requirement for PABP. Similarly, in lysates treated with anti-human eIF4G antibody, the cytosolic extract did not inhibit the translation of the chimeric reporter, thereby showing a requirement for eIF4G. These data show that translational silencing of Cp requires interactions of three essential elements of mRNA circularization, poly(A), PABP, and eIF4G. We suggest that Cp mRNA circularization brings the cytosolic Cp 3'-UTR-binding factor into the proximity of the translation initiation site, where it silences translation by an undetermined mechanism. These results suggest that in addition to its important function in increasing the efficiency of translation, transcript circularization may serve as an essential structural determinant for transcript-specific translational control.

Dual interactions of the translational repressor Paip2 with poly(A) binding protein.

The cap structure and the poly(A) tail of eukaryotic mRNAs act synergistically to enhance translation. This effect is mediated by a direct interaction of eukaryotic initiation factor 4G and poly(A) binding protein (PABP), which brings about circularization of the mRNA. Of the two recently identified PABP-interacting proteins, one, Paip1, stimulates translation, and the other, Paip2, which competes with Paip1 for binding to PABP, represses translation. Here we studied the Paip2-PABP interaction. Biacore data and far-Western analysis revealed that Paip2 contains two binding sites for PABP, one encompassing a 16-amino-acid stretch located in the C terminus and a second encompassing a larger central region. PABP also contains two binding regions for Paip2, one located in the RNA recognition motif (RRM) region and the other in the carboxy-terminal region. A two-to-one stoichiometry for binding of Paip2 to PABP with two independent K(d)s of 0.66 and 74 nM was determined. Thus, our data demonstrate that PABP and Paip2 could form a trimeric complex containing one PABP molecule and two Paip2 molecules. Significantly, only the central Paip2 fragment, which binds with high affinity to the PABP RRM region, inhibits PABP binding to poly(A) RNA and translation.

Poly(A)-binding protein interaction with elF4G stimulates picornavirus IRES-dependent translation.

The eukaryotic mRNA 3' poly(A) tail and the 5' cap cooperate to synergistically enhance translation. This interaction is mediated, at least in part, by elF4G, which bridges the mRNA termini by simultaneous binding the poly(A)-binding protein (PABP) and the cap-binding protein, elF4E. The poly(A) tail also stimulates translation from the internal ribosome binding sites (IRES) of a number of picornaviruses. elF4G is likely to mediate this translational stimulation through its direct interaction with the IRES. Here, we support this hypothesis by cleaving elF4G to separate the PABP-binding site from the portion that promotes internal initiation. elF4G cleavage abrogates the stimulatory effect of poly(A) tail on translation. In addition, translation in extracts in which elF4G is cleaved is resistant to inhibition by the PABP-binding protein 2 (Paip2). The elF4G cleavage-induced loss of the stimulatory effect of poly(A) on translation was mimicked by the addition of the C-terminal portion of elF4G. Thus, PABP stimulates picornavirus translation through its interaction with elF4G.

Interaction of eIF4G with poly(A)-binding protein stimulates translation and is critical for Xenopus oocyte maturation.

The poly(A)-binding protein Pab1p interacts directly with the eukaryotic translation initiation factor 4G (eIF4G) to facilitate translation initiation of polyadenylated mRNAs in yeast [1,2]. Although the eIF4G-PABP interaction has also been demonstrated in a mammalian system [3,4], its biological significance in vertebrates is unknown. In Xenopus oocytes, cytoplasmic polyadenylation of several mRNAs coincides with their translational activation and is critical for maturation [5-7]. Because the amount of PABP is very low in oocytes [8], it has been argued that the eIF4G-PABP interaction does not play a major role in translational activation during oocyte maturation. Also, overexpression of PABP in Xenopus oocytes has only a modest stimulatory effect on translation of polyadenylated mRNA and does not alter either the efficiency or the kinetics of progesterone-induced maturation [9]. Here, we report that the expression of an eIF4GI mutant defective in PABP binding in Xenopus oocytes reduces translation of polyadenylated mRNA and dramatically inhibits progesterone-induced maturation. Our results show that the eIF4G-PABP interaction is critical for translational control of maternal mRNAs during Xenopus development.

Serum-stimulated, rapamycin-sensitive phosphorylation sites in the eukaryotic translation initiation factor 4GI.

The eukaryotic translation initiation factor 4G (eIF4G) proteins play a critical role in the recruitment of the translational machinery to mRNA. The eIF4Gs are phosphoproteins. However, the location of the phosphorylation sites, how phosphorylation of these proteins is modulated and the identity of the intracellular signaling pathways regulating eIF4G phosphorylation have not been established. In this report, two-dimensional phosphopeptide mapping demonstrates that the phosphorylation state of specific eIF4GI residues is altered by serum and mitogens. Phosphopeptides resolved by this method were mapped to the C-terminal one-third of the protein. Mass spectrometry and mutational analyses identified the serum-stimulated phosphorylation sites in this region as serines 1108, 1148 and 1192. Phosphoinositide-3-kinase (PI3K) inhibitors and rapamycin, an inhibitor of the kinase FRAP/mTOR (FKBP12-rapamycin-associated protein/mammalian target of rapamycin), prevent the serum-induced phosphorylation of these residues. Finally, the phosphorylation state of N-terminally truncated eIF4GI proteins acquires resistance to kinase inhibitor treatment. These data suggest that the kinases phosphorylating serines 1108, 1148 and 1192 are not directly downstream of PI3K and FRAP/mTOR, but that the accessibility of the C-terminus to kinases is modulated by this pathway(s).

Eukaryotic translation initiation factor 4E (eIF4E) binding site and the middle one-third of eIF4GI constitute the core domain for cap-dependent translation, and the C-terminal one-third functions as a modulatory region.

The mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three roughly equal regions; an amino-terminal one-third (amino acids [aa] 1 to 634), which contains the poly(A) binding protein (PABP) and eIF4E binding sites; a middle third (aa 635 to 1039), which binds eIF4A and eIF3; and a carboxy-terminal third (aa 1040 to 1560), which harbors a second eIF4A binding site and a docking sequence for the Ser/Thr kinase Mnk1. Previous reports demonstrated that the middle one-third of eIF4GI is sufficient for cap-independent translation. To delineate the eIF4GI core sequence required for cap-dependent translation, various truncated versions of eIF4GI were examined in an in vitro ribosome binding assay with beta-globin mRNA. A sequence of 540 aa encompassing aa 550 to 1090, which contains the eIF4E binding site and the middle region of eIF4GI, is the minimal sequence required for cap-dependent translation. In agreement with this, a point mutation in eIF4GI which abolished eIF4A binding in the middle region completely inhibited ribosomal binding. However, the eIF4GI C-terminal third region, which does not have a counterpart in yeast, modulates the activity of the core sequence. When the eIF4A binding site in the C-terminal region of eIF4GI was mutated, ribosome binding was decreased three- to fourfold. These data indicate that the interaction of eIF4A with the middle region of eIF4GI is necessary for translation, whereas the interaction of eIF4A with the C-terminal region plays a modulatory role.

Eukaryotic translation initiation factor 4AIII (eIF4AIII) is functionally distinct from eIF4AI and eIF4AII.

Eukaryotic initiation factor 4A (eIF4A) is an RNA-dependent ATPase and ATP-dependent RNA helicase that is thought to melt the 5' proximal secondary structure of eukaryotic mRNAs to facilitate attachment of the 40S ribosomal subunit. eIF4A functions in a complex termed eIF4F with two other initiation factors (eIF4E and eIF4G). Two isoforms of eIF4A, eIF4AI and eIF4AII, which are encoded by two different genes, are functionally indistinguishable. A third member of the eIF4A family, eIF4AIII, whose human homolog exhibits 65% amino acid identity to human eIF4AI, has also been cloned from Xenopus and tobacco, but its function in translation has not been characterized. In this study, human eIF4AIII was characterized biochemically. While eIF4AIII, like eIF4AI, exhibits RNA-dependent ATPase activity and ATP-dependent RNA helicase activity, it fails to substitute for eIF4AI in an in vitro-reconstituted 40S ribosome binding assay. Instead, eIF4AIII inhibits translation in a reticulocyte lysate system. In addition, whereas eIF4AI binds independently to the middle and carboxy-terminal fragments of eIF4G, eIF4AIII binds to the middle fragment only. These functional differences between eIF4AI and eIF4AIII suggest that eIF4AIII might play an inhibitory role in translation under physiological conditions.

Trans-activation functions of the Sp-related nuclear factor, basic transcription element-binding protein, and progesterone receptor in endometrial epithelial cells.

The present study examined the trans-activation potential of basic transcription element-binding protein (BTEB), a recently identified member of the Sp family of GC box-binding transcription factors, on the expression of the gene encoding the pregnancy-associated, epithelial-specific, and progesterone (P)-induced porcine uterine endometrial secretory protein, uteroferrin (UF). Endometrial expression of BTEB, P receptor (PR), and UF genes was analyzed by RT-PCR as a function of pregnancy stage and cell type and was correlated with the levels of endometrial BTEB that were quantified by Western blot and/or electrophoretic mobility shift assay. PR, BTEB, and UF messenger RNAs (mRNAs) were present in early (day 12) and mid(day 60) pregnancy pig endometrium, although expression levels varied for each mRNA (UF, day 12 << day 60; PR and BTEB, day 12 = day 60). Within the endometrium, glandular epithelial (GE) cells manifested higher amounts of UF mRNA than stromal fibroblastic cells, whereas both cell types had comparable amounts of BTEB and PR mRNAs. Expression of BTEB, however, was limited to endometrial GE cells. A BTEB expression vector (pcDNA-3BTEB) was used to examine the effect of increased BTEB protein on UF gene expression and promoter activity in primary cultures of pig endometrial GE cells. Cells transiently transfected with pcDNA-3BTEB had 2-fold higher UF mRNA levels than those transfected with the empty expression vector (pcDNA-3). Further, cells cotransfected with a UF promoter-luciferase (-1935UF-Luc) reporter gene and the BTEB expression vector had 2-fold higher Luc activity than those cotransfected with reporter gene and pcDNA-3. This effect of BTEB was not observed in transfected endometrial stromal fibroblastic cells, but was apparent in the human endometrial epithelial carcinoma cell lines ECC-1 and Hec-1-A, which exhibit low levels of BTEB protein and low or undetectable PR mRNA levels, respectively. The respective contributions of BTEB and PR to the modulation of UF promoter activity were examined by cotransfection of Hec-1-A and ECC-1 cells with expression plasmids for BTEB and PR and one of two UF promoter constructs (-831UF-Luc or -1935UF-Luc) in the absence or presence of P. The increase in UF promoter activity with BTEB was mimicked by PR in a P-dependent manner in both cell lines. The combined effect of PR/P and BTEB appeared additive in Hec-1-A cells and was synergistic in ECC-1 cells. These results highlight the cell context dependence of the trans-activation potential of BTEB and suggest its unique role, in concert with PR, in directing the temporal expression of endometrial epithelial genes of pregnancy.

Eukaryotic initiation factor 4GII (eIF4GII), but not eIF4GI, cleavage correlates with inhibition of host cell protein synthesis after human rhinovirus infection.

For many members of the Picornaviridae family, infection of cells results in a shutoff of host protein synthesis. For rhinoviruses and enteroviruses, the shutoff has been explained in part by the cleavage of eukaryotic initiation factor 4GI (eIF4GI), a component of the cap-binding protein complex eIF4F. The cleavage of eIF4GI is mediated by the virus-specific proteinase 2Apro and results in inhibition of cap-dependent, but not cap-independent, translation. The inhibition of host protein synthesis after infection with human rhinovirus 14 (HRV-14) lags behind the cleavage of eIF4GI. Recently, we discovered a functional homolog of eIF4GI, termed eIF4GII, and showed that cleavage of eIF4GII coincides with the shutoff of host cell protein synthesis after poliovirus infection (Gradi et al., Proc. Natl. Acad. Sci. USA 95:11089-11094, 1998). We wished to determine whether eIF4GII cleavage kinetics could also explain the lack of correlation between the kinetics of eIF4GI cleavage and the shutoff of host protein synthesis after rhinovirus infection. In this study, we examined the correlation between human rhinovirus-induced shutoff of host protein synthesis and cleavage of eIF4GI and eIF4GII. In HRV-14-infected HeLa cells, almost no intact eIF4GI could be detected by 4 h postinfection, while only 4% of eIF4GII was cleaved at this time. By 6 h, however, 67% of eIF4GII was cleaved, and this cleavage coincided with a significant (60%) decline of host translation. These results suggest that cleavage of both eIF4GI and eIF4GII is required for HRV-mediated inhibition of host cell protein synthesis and that the cleavage of eIF4GII is the rate-limiting step in the shutoff of host cell protein synthesis after rhinovirus infection.