mRNA Translation without eIFs Figure 1. Requirement of eIF4G for translation of C+Luc sg-mRNA. Panel A. Protein synthesis and eIF4G cleavage in BHK cells. BHK cells were electroporated with 30 mg of IRES-2A mRNA or transcription buffer as a control. At 2 hpe, cells were again electroporated with 20 mg of C+Luc mRNA or cap-Luc-poly(A) or IRES-Luc-poly(A) as mRNAs control (mRNAs are schematized in the upper part of each figure). Values of luc activity obtained from the different mRNAs are represented. eIF4GI and eIF4GII integrity was analyzed by western blot analysis of extracts recovered at 2 and 4 hpe of IRES-2A. Tubulin was also analyzed to adjust the amount of each sample loaded onto the gel. Panel B. Translation directed by several mRNAs after the cleavage of eIF4G in HeLa S3 extracts. Translation was carried out in Hela S3 extracts pre-treated with purified MBP-2A or MBP proteins. Luc production was determined by measuring luc activity from each translation mixture and the integrity of eIF4GI and eIF4GII was analyzed by western blotting. Panel C. Effect of eIF4G cleavage on protein synthesis in BHK cells transfected with different SV replicons. BHK cells were electroporated with 20 mg of rep C+Luc mRNA (Schematized in the figure). At 2 hpe, cells were again electroporated with 30 mg of IRES-2A mRNA or transcription buffer. At 1, 3 and 5 hpe of IRES-2A mRNA, cell cultures were harvested and luc activity was measured or examined by western blotting with anti-luc antibodies (left panel). eIF4GI and eIF4GII integrity was tested by western-blot analysis. Tubulin was also analyzed to adjust the amount of each sample loaded onto the gel (right panel). a-Tub: a-Tubulin; luc: luciferase; C-t: C-terminal cleavage product of eIF4G; N-t: N-terminal cleavage product of eIF4G. doi:10.1371/journal.pone.0004772.g001 (Fig 2C, upper panel) whereas, as shown above, this compound had little effect in cells electroporated with rep C+Luc (Fig 2B, upper panel). As a control, translation of a cap-Luc-poly(A) mRNA was strongly inhibited by arsenite as expected. The phosphorylation of eIF2a was analyzed in parallel (Fig 2C, lower panel). Arsenite treatment induced a dose dependent increase in the amount of phosphorylated eIF2a, while the unphosphorylated form dimin- ished. Therefore, this phosphorylation induced by arsenite correlates with the inhibition of C+Luc and cap-Luc-poly(A) translation. Also, the action of dithiothreitol, another compound that induces eIF2a phosphorylation, was tested and the results were similar to those described for arsenite (results not shown). In principle, the structure of C+Luc mRNA synthesized in vitro is similar to that produced by the SV transcriptional machinery, although the interactions of this mRNA with cellular proteins may vary according to whether it is directly electroporated into cells or synthesized inside BHK cells. To explore the effect of arsenite on the translational behaviour of C+Luc and cap Luc mRNAs transcribed in the nucleus, BHK cells were transfected with the pcDNA C+Luc or pcDNA Luc plasmids, which are substrates for RNA pol II that produce C+Luc and Luc mRNAs, respectively. At 18 hpe cultures were labelled with [35S]-Met/Cys in absence or presence of arsenite and luc was immunoprecipitated with anti-luc antibodies (Fig 2D). Arsenite treatment inhibits cellular translation (Fig 2D, upper panel) as well as luc synthesis (Fig 2D, lower panel). No differences exist between the inhibition by arsenite and luc synthesis from C+Luc or Luc mRNAs. Moreover, translation in HeLa S3 cell extracts was also assayed after the addition of poly PLoS ONE | www.plosone.org 3 March 2009 | Volume 4 | Issue 3 | e4772