8048 Nucleic Acids Research, 2010, Vol. 38, No. 22 Figure 6. Speciﬁc PUF protein 4E-BP complexes are isolated from cells. Afﬁnity chromatography purifying indicated PUF–TAP complexes from cells bearing the indicated TAP-tagged PUF protein or an untagged control and Myc tagged (A) Caf20p or (B) Eap1p. Upper panels probed with anti-protein A (aTAP) and lower with anti-Myc antibodies. I, input [10 mg total protein, P = Pellet (0.5mg) S = supernatant (10 mg unbound fraction)]. The immunoblots are representative of three independent experiments. Myc-tagged EAP1 and repeated the TAP-afﬁnity chromatography experiments to identify whether any Eap1p-PUF complexes formed (Figure 6B). Remarkably these experiments revealed a complementary set of inter- actions, as a fraction of Eap1p was associated with Puf1p and Puf2p, but not the other PUFs tested. As each set of experiments identiﬁed different protein complexes it provides evidence that they are not artifactual. Because the Protein A component of the TAP tag bound our Myc antibodies, we were not able to complete reciprocal precipitation experiments. Instead, we made new strains bearing Myc-tagged PUF proteins (PUF2, PUF3 and PUF5) and TAP tagged 4E-BPs and repeated the TAP-afﬁnity chromatography experiments. In agree- ment with our original experiment, a small fraction of Puf5p-Myc was associated with Caf20p-TAP and Puf2p-Myc with Eap1p-TAP (Supplementary Figure S2). Importantly, no associations were seen between Puf3p-Myc and either 4E-BP-TAP protein or between Myc-tagged proteins and the protein A resin in control strains lacking a TAP tag, although a tiny fraction of Puf2p-Myc was additionally found associated with Caf20p-TAP. These experimental data do not entirely agree with the in silico predictions. The predicted Caf20p-Puf5p inter- action was found. However the Caf20p-Puf4p and Eap1p-Puf1/2p interactions we identiﬁed were not pre- dicted by our statistical comparisons. There are several possible explanations for this including the fact that our statistical treatment did not take into consideration mRNA abundances or the relative afﬁnities of the inter- actions, as these are not known. Our experiments repre- sent a ‘snap-shot’ of cells from non-stressed log cultures. Under different growth stresses, phases or synchronous states the associations may differ. However our data has shown that there is value using this type of approach to devise hypotheses to test experimentally. Importantly, when taken together, these experiments indicate that each yeast 4E-BP can form mRNA–protein complexes with speciﬁc PUF-bound mRNAs. In summary, by using a translational proﬁling approach, we have identiﬁed that translation of a signiﬁ- cant fraction of yeast mRNAs is regulated by the yeast eIF4E-BPs Caf20p and Eap1p. Many of the regulated transcripts are poorly translated in wild-type cells during exponential growth and these mRNAs are enriched in function in gene expression, cell cycle or transport. In addition to explore how mRNA speciﬁcity is achieved, we provide direct evidence that each yeast 4E-BP can interact with speciﬁc PUF proteins. As PUF proteins bind RNAs with sequence speciﬁcity, this could present one mechanism by which the yeast 4E-BPs target individ- ual mRNAs. By analogy with higher eukaryotes, it is likely that these complexes contain additional components that remain to be determined and that other 30-UTR-binding protein/4E-BP combinations also await identiﬁcation. These studies, therefore, provide the ﬁrst evidence that the yeast 4E-BPs regulate translation of hundreds of mRNAs and that by further study it will be possible to uncover molecular details in yeast that inform studies in higher eukaryotes. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank the Microarray facility at the University of Manchester (especially A. Hayes and L. Wardleworth)