available at ArrayExpress (www.ebi.ac.uk/microarray/ using accession numbers caf20D: E-MEXP-1308; eap1D: E-MEXP-1309). Quantitative RT–PCR analysis RNA analysis by RT–PCR was carried out using the MyIQ single-colour real-time PCR detection system and IQ SYBR Green Supermix (BioRad Laboratories) as described earlier (21,22). Signals were quantiﬁed relative to actin mRNA control. TAP afﬁnity chromatography PUF-TAP or 4E-BP-TAP and control strains were grown in YPD medium to a concentration of 6 106 cells per ml. Cultures were harvested by centrifugation (5500g, 10 min, 4 C), washed in ice cold 1 mM phenylmethlysulphonyl ﬂuoride (PMSF) and resuspended in two cell volumes of Buffer A [20 mM Tris–HCl (pH 8.0)], 140 mM KCl, 1.8 mM MgCl2, 0.1% Nonidet P-40 (NP-40), 0.5 mM dithiothreitol (DTT), 1 mM PMSF, 1 complete EDTA-free protease inhibitor cocktail tablet [Roche], 100 U/ml RNasin [Promega]. Cell suspension was frozen and ground under liquid nitrogen and stored at 80 C. Crude cell lysates were cleared by centrifugation (5500g for 20 min, 4 C). Extracts (2 mg) were incubated with 400 ml (50% [v/v]) IgG Sepharose beads (GE Healthcare) for 2 h at 4 C and afﬁnity puriﬁed as described earlier (19). PUF proteins were released from the IgG beads by heating at 65 C for 3 min in 2 non-reducing Protein Loading Buffer [62.5 mM Tris–HCl (pH 6.8), 2% SDS, 10% (v/v) glycerol, 0.002% (w/v) bromphenol blue]. Immunoblotting Whole cell extracts; Cells were harvested and resuspended in three times wet pellet volume of Buffer B [50 mM Tris– HCl pH 7.5, 100 mM NaCl 1 mM EDTA 1 mM PMSF and 1 Protease Inhibitors (Roche)] and ground in liquid Nitrogen. Samples were assayed for protein concen- tration (Bradford) and diluted to 1 ug protein per ml. TAP proteins were resolved by SDS–PAGE electrophoresis followed by transfer to a nitrocellulose membrane. Immunoblotting detection used horseradish peroxidase (HRP) conjugated primary antibodies to Protein A (Abcam) and c-Myc (9E10, Santa Cruz Biotechnology and 4A6, Millipore), and chemiluminescent detection (Pierce Biotechnology). Other primary antibodies used were caf20p (10), Gcd11p (23), Cic1p (24), Lsm8p (25), Taf3p, Taf7p (26), Sec9p (27), Pub1p (28), Ade2p and Arp2p (Santa Cruz Biotechnology) and were detected using appropriate HRP-conjugated secondary antibodies as indicated earlier. RESULTS AND DISCUSSION Nucleic Acids Research, 2010, Vol. 38, No. 22 8041 could be surveyed simultaneously. We used isogenic strains produced by the systematic yeast gene deletion project for this study. Our initial growth characterization revealed that the caf20D and wild-type strains behaved identically in both rich and minimal medium, while the eap1D strain typically exhibited an initial growth lag when recovering from stationary phase, but then proliferated at a rate indistinguishable from the wild-type strain (data not shown). Polyribosome proﬁle analysis revealed a minor halfmer phenotype for the eap1D strain, but no obvious global defect in the caf20D strain (Figure 1A). For array analysis the following samples were collected: (i) total RNA samples to control for changes in transcript abundance and (ii) polysome gradient fractionated samples. Fractionated samples were split into monosomal and polysomal fractions as indicated in Figure 1A. Each RNA sample was processed into complementary RNA and hybridized to Affymetrix yeast S98 gene chip arrays using standard protocols (21,22). All analyses were performed in duplicate and the resulting data were analysed and compared using standard bioinformatics procedures (as outlined in the Supplementary Data). MA plots and statistical analysis of variance tests (Supplementary Figure S1A, B, D, E, G and H and data not shown) indicated that there was only modest dispersal between biological replicate samples, conﬁrming that the arrays are reliable reporters of relative transcript abundance in our samples. As anticipated, a greater dispersal of data points was seen between wild-type and mutant samples (Supplementary Figure S1C, F and I). 4E-BPs have minimal impact on mRNA abundance Under normal cellular conditions, changes in mRNA transcript abundance reﬂect changes in both mRNA syn- thesis and decay. In this study, overall transcript abun- dance changes were relatively minor. caf20D alters transcript abundance >2-fold for 100 genes (29 up and 71 down) and eap1D 99 genes (56 up and 43 down). A complete listing of signiﬁcantly altered genes is shown in Supplementary Tables S1–S4. As expected the transcript signals for the deleted gene were down-regulated most in each data set. The small number of genes affected is con- sistent with 4E-BPs having no widespread direct transcrip- tional role, instead the changes observed could be an indirect consequence of altered translation of transcrip- tion factors (see below). There was also no evidence of ‘potentiation’. This is a term used to describe co-ordinated transcription and translation responses observed during the reprogramming of gene expression to adapt to some cellular stresses (21,29). Fewer than 10 genes were both transcriptionally and translationally up-/down-regulated across both experiments (Figure 1F; numbers in black ﬁlled circles). Microarray approach to determine roles for 4E-BPs Overview of translational controls To determine whether translation of speciﬁc mRNAs was altered by elimination of Caf20p or Eap1p under steady state growth conditions, we used a microarray-based approach so that the translation state of all mRNAs We measured polysome:monosome [P/M] ratios for each transcript (probe set), termed a ‘translational state’, calculated as log2[P/M] ratio for each mutant and wild-type control from mean normalized sample