Multiple isoforms of the translation initiation factor eIF4GII are generated via use of alternative promoters, splice sites and a non-canonical initiation codon.

During the initiation stage of eukaryotic mRNA translation, the eIF4G (eukaryotic initiation factor 4G) proteins act as an aggregation point for recruiting the small ribosomal subunit to an mRNA. We previously used RNAi (RNA interference) to reduce expression of endogenous eIF4GI proteins, resulting in reduced protein synthesis rates and alterations in the morphology of cells. Expression of EIF4G1 cDNAs, encoding different isoforms (f-a) which arise through selection of alternative initiation codons, rescued translation to different extents. Furthermore, overexpression of the eIF4GII paralogue in the eIF4GI-knockdown background was unable to restore translation to the same extent as eIF4GIf/e isoforms, suggesting that translation events governed by this protein are different. In the present study we show that multiple isoforms of eIF4GII exist in mammalian cells, arising from multiple promoters and alternative splicing events, and have identified a non-canonical CUG initiation codon which extends the eIF4GII N-terminus. We further show that the rescue of translation in eIF4GI/eIF4GII double-knockdown cells by our novel isoforms of eIF4GII is as robust as that observed with either eIF4GIf or eIF4GIe, and more than that observed with the original eIF4GII. As the novel eIF4GII sequence diverges from eIF4GI, these data suggest that the eIF4GII N-terminus plays an alternative role in initiation factor assembly.

Localization of ribosomes and translation initiation factors to talin/beta3-integrin-enriched adhesion complexes in spreading and migrating mammalian cells.

BACKGROUND INFORMATION: The spatial localization of translation can facilitate the enrichment of proteins at their sites of function while also ensuring that proteins are expressed in the proximity of their cognate binding partners. RESULTS: Using human embryonic lung fibroblasts and employing confocal imaging and biochemical fractionation techniques, we show that ribosomes, translation initiation factors and specific RNA-binding proteins localize to nascent focal complexes along the distal edge of migrating lamellipodia. 40S ribosomal subunits appear to associate preferentially with beta3 integrin in focal adhesions at the leading edges of spreading cells, with this association strongly augmented by a synergistic effect of cell engagement with a mixture of extracellular matrix proteins. However, both ribosome and initiation factor localizations do not require de novo protein synthesis. CONCLUSIONS: Taken together, these findings demonstrate that repression, complex post-transcriptional regulation and modulation of mRNA stability could potentially be taking place along the distal edge of migrating lamellipodia.

Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: a reappraisal of translation initiation during mitosis.

Translation mechanisms at different stages of the cell cycle have been studied for many years, resulting in the dogma that translation rates are slowed during mitosis, with cap-independent translation mechanisms favored to give expression of key regulatory proteins. However, such cell culture studies involve synchronization using harsh methods, which may in themselves stress cells and affect protein synthesis rates. One such commonly used chemical is the microtubule de-polymerization agent, nocodazole, which arrests cells in mitosis and has been used to demonstrate that translation rates are strongly reduced (down to 30% of that of asynchronous cells). Using synchronized HeLa cells released from a double thymidine block (G 1/S boundary) or the Cdk1 inhibitor, RO3306 (G 2/M boundary), we have systematically re-addressed this dogma. Using FACS analysis and pulse labeling of proteins with labeled methionine, we now show that translation rates do not slow as cells enter mitosis. This study is complemented by studies employing confocal microscopy, which show enrichment of translation initiation factors at the microtubule organizing centers, mitotic spindle, and midbody structure during the final steps of cytokinesis, suggesting that translation is maintained during mitosis. Furthermore, we show that inhibition of translation in response to extended times of exposure to nocodazole reflects increased eIF2α phosphorylation, disaggregation of polysomes, and hyperphosphorylation of selected initiation factors, including novel Cdk1-dependent N-terminal phosphorylation of eIF4GII. Our work suggests that effects on translation in nocodazole-arrested cells might be related to those of the treatment used to synchronize cells rather than cell cycle status.

Inhibition of mammalian target of rapamycin (mTOR) signalling in C2C12 myoblasts prevents myogenic differentiation without affecting the hyperphosphorylation of 4E-BP1.

Current accepted models suggest that hypophosphorylated 4E-binding protein (4E-BP1) binds to initiation factor 4E (eIF4E) to inhibit cap-dependent translation, a process readily reversed by its phosphorylation following activation of mammalian target of rapamycin (mTORC1) signalling. Myogenic differentiation in the C2C12 myoblast model system reflects a concerted and controlled activation of transcription and translation following the exit of cells from the cell cycle. Here we show that myogenic differentiation is associated with increased rates of translation, the up-regulation of both 4E-BP1 mRNA and protein levels and enhanced levels of eIF4E/4E-BP1 complex. Paradoxically, treatment of C2C12 myoblasts with an inhibitor of mTOR signalling (RAD001) which inhibits translation, promotes the hyperphosphorylation of 4E-BP1 on novel sites and prevents the increase in 4E-BP1 levels. In contrast, eIF4E appears to be under translational control with a significant delay between induction of mRNA and subsequent protein expression.

Human rhinovirus type 89 variants use heparan sulfate proteoglycan for cell attachment.

We have previously isolated mutants of the major-group human rhinovirus type 89 that grow in cells deficient in intercellular adhesion molecule 1 (ICAM-1), the receptor used by the wild-type virus for cell entry [A. Reischl, M. Reithmayer, G. Winsauer, R. Moser, I. Goesler, and D. Blaas., J. Virol. 75:9312-9319, 2001]. We now demonstrate that one of these variants utilizes heparan sulfate proteoglycan (HSPG) as a cellular receptor. Adaptation to ICAM-1-deficient cells not only resulted in the newly acquired receptor specificity but also rendered the virus less stable at low pH and at elevated temperatures. This instability might compensate for the absence of the uncoating activity of ICAM-1. Whereas wild-type virus infection via ICAM-1 proceeded in the presence of the vesicular H(+)-ATPase inhibitor bafilomycin A1, infection by the mutant via HSPG was prevented by the drug. This suggests that the low pH prevailing in endosomal compartments is required for uncoating in the absence of the catalytic activity of ICAM-1.