Figure 5. Translation state change for Puf1-5p associated mRNAs in caf20D and eap1D cells. A comparison of the change in translation state for mRNAs identiﬁed by Gerber and colleagues as bound to Puf1p (35 mRNAs), Puf2p (142), Puf3p (219), Puf4p (202) and Puf5p (203 mRNAs) is plotted (open circle, open rectangle) for caf20D and eap1D, respectively. The median (grey horizontal bar) and upper and lower quartiles are indicated by the box-plot. Statistical analyses are shown in Supplementary Table S9. proteins were also seen but only Puf4p showed a statistic- ally signiﬁcant enrichment (Supplementary Table S9). However, when plotted graphically (Figure 5) the mean distributions were not shifted >0, suggesting that only a few Puf-associated transcripts are potentially translation- ally regulated by Eap1p. A more recent RIp-chip study has identiﬁed mRNAs associated with a wide range of RNA-binding proteins (39). Equivalent comparative statistical analyses revealed potential overlap between caf20D up-regulated genes and mRNA-binding proteins including translational regula- tors (Khd1p, Mrn1p, Scp160p), factors involved in nuclear export of mRNA (Gbp2 and Nab2p) as well as Pub1p and Ssd1p. Similar comparisons with our eap1D data revealed overlaps with Nab2, Nrd1p, and the cyto- plasmic polyA binding protein Pab1p (Supplementary Table S9). In addition, we found other relationships between the data sets, including correlations between mRNAs bound by speciﬁc proteins and those translation- ally down-regulated by the 4E-BP deletions. For example Mrn1p and Pub1p-bound mRNAs are both enriched among mRNAs down-regulated by eap1D and up- regulated by caf20D. We also noted that there were examples where fewer than expected mRNAs in common between speciﬁc data sets (Supplementary Table S9). While not conclusive, these comparisons suggest that different proteins binding to speciﬁc mRNAs could combine with 4E-BPs to regulate their translation in yeast in a manner similar to that proposed for multicellular eukaryotes. Because there are several yeast PUF proteins, we decided to examine experimentally whether there were any interactions between the 4E-BPs Nucleic Acids Research, 2010, Vol. 38, No. 22 8047 and the PUF proteins that could help to explain the polysome microarray data. 4E-BPs physically associate with speciﬁc PUFs PUF proteins are found in diverse eukaryotes (as are 4E-BPs) and have been implicated in post-transcriptional control of gene expression in early development, germ cells and neuronal cells (40,41). PUFs have been implicated both in the control of mRNA stability and translational repression (40). Unlike the 4E-BPs, PUFs are sequence speciﬁc RNA-binding proteins that contain eight repeats of an 40 residue motif, the PUF motif (42). PUF proteins often associate with 30-UTRs (19) where they can form complexes with other proteins (43). In yeast, Pufs 1 and 2 preferentially bind mRNAs encoding membrane-associated proteins, Puf4p rRNA-processing factor mRNAs and Puf5p chromatin modiﬁer mRNAs (19). Puf6p was found to bind the ASH1 30-UTR and repress its translation in mother cells. ASH1 mRNA is normally localized to the distal bud tip and both mRNA localization and translation were affected in puf6D cells (44). Most studies to date point to a function in mRNA stability, with each PUF protein having distinct targets, although few mRNA targets have been studied in any detail. Puf3p preferentially binds mitochondrial-localized transcripts e.g. COX17 to stimulate the latter’s degrad- ation (45,46). In another study, the stability of 38 potential PUF-target mRNAs was assessed and only two of these (HXK1 and TIF1) were found to have altered mRNA half-lives in pufD strains under the conditions studied (47). One favourable explanation for a lack of effects on mRNA stability for certain targets is that PUF protein binding can mediate translational control. In support of this idea, the Wickens group has shown that both Puf4p and Puf5p bind HO mRNA and regulate post- transcriptional processes including mRNA stability and translational regulation in vitro (48,49). Intriguingly, we found that HO polysome association is signiﬁcantly up-regulated in caf20D cells (Supplementary Table S5). These observations combined with our statistical analysis suggested to us that speciﬁc Caf20p-PUF inter- actions may determine in part which mRNAs are transla- tionally regulated by 4E-BPs. To determine if any of the statistical observations re- ﬂected real interactions, we obtained TAP-tagged PUF strains and incorporated tandem Myc epitopes at the C-terminus of CAF20 in each. Afﬁnity chromatography was performed on each TAP strain and an untagged control in the presence of RNase inhibitors but in the absence of any crosslinker, as described (19) and the reac- tions were probed with anti-TAP and anti-Myc sera. Figure 6A shows that in each case the TAP-tagged protein was efﬁciently captured by this procedure. We found that a portion of Caf20p was associated with TAP-tagged Puf5p and to a lesser extent Puf4p, while none was associated with Pufs1-3 or the untagged control. These associations required RNA because they were not identiﬁed when RNase inhibitors were omitted from the buffers used (data not shown). We made an equivalent set of strains bearing C-terminally