on the left in Figure 1E, the bias is less pronounced, suggesting distinct roles for Caf20p and Eap1p in re- pressing translation of genes under normal growth conditions. As an independent conﬁrmation that the arrays accur- ately reported translation state changes, quantitative RT– PCR analysis was performed on polysome fractionated RNA samples with oligonucleotide primers speciﬁc for a subset of genes (Figure 2A). Genes were selected to cover a range of up- and down-regulated mRNAs and also some unregulated mRNAs. Despite differences between these techniques, a good correlation was observed between the array and RT–PCR approaches. The 4E-BPs interact with eIF4E via a common motif, and eIF4E itself binds the cap structure common to all mRNAs. Therefore it seems unlikely that these features alone impart the observed mRNA speciﬁc translational controls. Therefore our data suggest that there must be other factors, which work in concert with the yeast 4E-BPs to mediate control of translation (see below). It seemed likely to us that these other factors may bind to speciﬁc sequences within each mRNA and contribute to the observed control. As such, it was important to verify whether protein levels vary as anticipated by the transla- tional proﬁling data. Almost all efﬁcient strategies for epitope-tagging yeast genes involve disrupting either the 30-UTR or replacing elements 50 of the ORF. The 50- and 30-UTRs are common sites used by RNA-binding factors to mediate mRNA speciﬁc controls in many eukaryotes, so we reasoned that using a tagging approach was not appropriate here. Instead we searched the literature to identify available protein speciﬁc antibodies. We screened the antibodies available and identiﬁed several that functioned with our whole-cell extracts. The antibodies were used and compared with an actin loading control that did not vary between strains (Figure 2B). Antibody signals varied between the strains tested in a manner that mirrored the trends seen in polysome/monosome ratios from our arrays. Thus Taf7p levels varied and were increased in caf20D and decreased in eap1D while both Cic1p and Lsm8p were more abundant in eap1D cells. Thus our arrays do appear to reﬂect the translational controls—at least for those candi- dates independently veriﬁed by RT–PCR and/or western blotting. This includes CIC1 and TAF3, which are both up-regulated translationally by eap1D in our microarrays, by RT–PCR and western blotting. An exception is Pub1p where RT–PCR conﬁrmed the microarray data suggesting enhanced PUB1 polysome association in caf20D cells, but steady state protein levels were not signiﬁcantly altered in this strain. These data suggest additional factors act to control Pub1p levels. A signiﬁcant overlap between caf20 and eap1 data sets Although the majority of mRNA targets differ between the array experiments, we observed a statistically signiﬁ- cant overlap between the mRNAs similarly up-regulated by both eap1D and caf20D (52 genes P = 9.8 10 23; Figure 1D–F, Supplementary Table S9). In contrast, there are very few reciprocally regulated genes (e.g. only Nucleic Acids Research, 2010, Vol. 38, No. 22 8043 four genes up-regulated by caf20D are down-regulated by eap1D; Figure 1F). During the course of our analyses, immunoblotting with anti-Caf20p anti-serum revealed that the steady state expression levels of Caf20p are reduced in eap1D cells (data not shown). As this observa- tion may explain the observed overlap in translational control between these factors, we examined carefully the expression levels of both 4E-BPs. Myc-epitope tags were added by homologous recombination to the C-termini of CAF20 and EAP1 in separate strains. Immunoblotting conﬁrmed that Caf20p expression was reduced 50% in eap1D cells, while Eap1p levels were maintained in caf20D cells (Figure 1G). Therefore the observed reduced Caf20p levels may cause lower afﬁnity Caf20p target mRNAs to lose normal translational control in eap1D cells and contribute to the overlap in array data sets. 4E-BPs repress genes controlling gene expression To gain insight into the functions of the translationally regulated transcripts, each regulated gene was placed into a functional class based upon classiﬁcations included within the GOslim mapper (Saccharomyces Genome Database) and annotations at the Yeast Proteome Database (Figure 3 and Supplementary Tables S1–S8 and S10). This analysis revealed that many tran- scripts with enhanced [P/M] ratios are themselves involved in gene expression. Deletion of either 4E-BP enhances translation of many transcription factors and chromatin remodelling enzymes. These changes did not cause large changes in transcript abundance as arrays of total mRNA showed that few genes are altered by >2-fold (Supplementary Tables S1–S4). We assume that these cells have adapted to the loss of each factor and that the changes to the translation proﬁle observed have normalized overall transcription as far as possible under the standard growth conditions examined. RNA metabol- ism is another abundant class of up-regulated genes for both 4E-BPs. In addition, Eap1p represses translation of many ribosome biogenesis factors involved in synthesis/ assembly of both ribosomal subunits. This may account for the apparent modest halfmer phenotype observed in eap1D cells (arrowed in Figure 1A). Overall these data suggest that regulation of genes involved in RNA metab- olism is an important function of these 4E-BPs and that each protein regulates speciﬁc targets involved in the same broad pathways/processes. Caf20p represses translation of CLN genes and those important for polarised growth The cell cycle, budding and site of polarised growth GO Slim categories are particularly over-represented within the translationally up-regulated caf20D mRNAs. Cyclins CLN1, CLN2, CLN3, CLB1, and CLB4 were among many cell cycle associated mRNAs. As indicated in the introduction, one of the best-characterized examples of a gene regulated by eIF4E availability in yeast is the G1 cyclin CLN3. A temperature sensitive mutation in yeast eIF4E (cdc33-1) has a G1-S phase cell cycle defect that is overcome by elevated expression of CLN3 (14). We inter- pret our array data as conﬁrming the importance of