8046 Nucleic Acids Research, 2010, Vol. 38, No. 22 Figure 4. eap1D and caf20D cells have nitrogen utilization defects. Cells complemented for auxotrophic markers were grown in SC complete medium to A600 = 0.6 washed and diluted to A600 = 0.1, then 10-fold serially diluted and spotted (3 ml) onto the indicated media. Growth was scored on a scale of 0 (none), +/ (minimal) to 3+ (wild-type). Table 1. Maximum doubling times of strains in liquid medium with indicated nitrogen sources Strain Wild-type Caf20D eap1D Complete 118 ± 2a 124 ± 5 140 ± 15 Medium Serine 148 ± 20 182 ± 20 212 ± 19 Glutamate 153 ± 10 189+21 233 ± 35 aMean maximum doubling times in min ± SD (n = 9 cultures) for cultures grown in liquid media equivalent to solid media shown in Figure 4. growth on alternate nitrogen sources, while a plasmid bearing missense mutations that disrupt the eIF4E inter- action motif in Eap1p (EAP1-mt in Figure 4) shown to eliminate eIF4E binding (10) was signiﬁcantly impaired in this response, suggesting that eIF4E-binding contributes to this phenotype. These data show that 4E-BP-mediated translational controls are important for normal amino acid metabolism in yeast in addition to their role in pseudohyphal development in 1278b yeast cells (10). Overlap between 4E-BP regulated and PUF-bound mRNAs One paradigm for the action of 4E-BPs is that translation- al repression is mediated in part via protein–protein inter- actions between proteins bound to the 50-cap and those attached to the 30-UTR of speciﬁc mRNAs (2). As our array data reveal that Eap1p and Caf20p largely regulate translation of distinct genes (Figure 1), it seemed likely that additional mRNA-binding factors may contribute to the control mechanism. Several studies have used microarrays to identify RNAs associated with speciﬁc RNA-binding proteins following immune-precipitation of epitope tagged proteins (RIp-chip). We used statistical comparisons to assess if there were any signiﬁcant overlaps between the identities of our 4E-BP regulated transcripts and transcripts identiﬁed by others as speciﬁcally associated with a range of mRNA-binding proteins in different RIp-chip experiments (19,37–39). The question addressed was: are genes identiﬁed as enriched with speciﬁc mRNA binding proteins also those identiﬁed as translationally controlled by the 4E-BPs or are the associations random? This analysis considers each transcript equally and does not account for mRNA abundance or any biases in the data sets. We found that there was a potentially signiﬁcant overlap between genes up-regulated by caf20D (i.e. trans- lation state change >0.9) and mRNAs associated with Puf1p, Puf2p, and Puf5p (P 0.0001) (Supplementary Table S9). The full distribution is shown graphically in Figure 5, where the translational state change for each PUF-bound mRNA is shown for the caf20D data. This additionally shows that the mean translational states for Puf1p, Puf2p and Puf5p are each shifted positively away from zero, while the means for Puf3p and Puf4p are not. As Gerber and colleagues found fewer mRNAs associated with Puf1p and Puf2p than Puf3-5p, the apparent enrich- ment in our caf20D up-regulated genes associated with Puf5p appeared most signiﬁcant. Overlaps between genes translationally up-regulated by eap1D and bound by PUF