8040 Nucleic Acids Research, 2010, Vol. 38, No. 22 regulated signalling events activate both cytoplasmic polyadenylation, disrupt the Maskin-eIF4E interaction and promote translation (2,5). Saccharomyces cerevisiae possesses two 4E-BPs, Caf20p and Eap1p of 18 and 70 KDa, respectively. Neither protein shares obvious sequence homology with each other or 4E-BPs from non-yeast species, except for the eIF4E interaction consensus motif, however both yeast 4E-BPs interact with eIF4E to inhibit translation of capped reporter mRNAs (6,7). Although deletion of either 4E-BP gene confers no growth defect under standard laboratory conditions, caf20D alleviates growth defects of some translation factor mutations and converse- ly its overexpression exacerbates these phenotypes (8). eap1D causes rapamycin resistance (7) and partial resist- ance to translation inhibition caused by diamide (a thiol oxidant), and cadmium (a heavy metal) (9). In the yeast 1278b background deletion of either 4E-BP prevents pseudohyphal growth following nitrogen limitation (10). Similarly, eap1D and caf20D cells respond differently to membrane stress (11,12). Taken together the data are con- sistent with the idea that each 4E-BP interacts with eIF4E and competes with eIF4G to modulate the translation of a different subset of speciﬁc yeast genes. Little progress has been made in identifying speciﬁc mRNA targets regulated by the yeast 4E-PBs. One possible candidate is the G1 cyclin CLN3 as it can be controlled by altering eIF4E activity. CLN3 mRNA contains a short up-stream ORF that plays a role repress- ing Cln3p expression (13). Temperature sensitive eIF4E mutations (e.g. cdc33-1) confer G1 arrest and reduce ex- pression of CLN3 and enhanced CLN3 expression is suf- ﬁcient to restore G1-S phase progression (14). Recently, a second mRNA target, POM34 mRNA was described, which encodes a membrane protein component of the nuclear pore complex. In cells bearing spindle-pole body duplication defects Eap1p was required for POM34 trans- lation (15). Given the paucity of information concerning speciﬁc 4E-BP targets, we used a translational proﬁling approach to identify mRNAs whose translation is altered following loss of each yeast 4E-BP and found that both altered translation of a large fraction of yeast genes. Quantitative real-time reverse transcription- polymerase chain reaction (RT–PCR) and immunoblot analyses have validated the micro-array approach for selected targets and a phenotypic analysis indicates that the observed altered translation of several nitrogen metab- olism genes may contribute to the sensitivity of the eap1D strain to growth on some nitrogen sources. Despite sharing a common interaction site on eIF4E, we ﬁnd that most target mRNAs differ between the 4E-BPs sug- gesting that additional factors are necessary to impart 4E-BP mRNA speciﬁcity. To begin to address this mech- anistic issue we compared the identities of mRNAs found by others to interact with speciﬁc RNA-interacting proteins, with our translational proﬁling data. Our analysis suggested that certain yeast PUF proteins may be among those important for mRNA speciﬁcity. As PUF proteins have been implicated in translational control in higher organisms, we tested this prediction directly and report that Puf4/5p-bound mRNA complexes associate with a fraction of Caf20p, while Puf1/2p associ- ate with Eap1p. Taken together these studies reveal that 1000 genes are subject to translational control via the 4E-BPs and provides evidence that 4E-BP association with speciﬁc 30-UTR-binding proteins imparts mRNA target speciﬁcity in yeast. MATERIALS AND METHODS Yeast strains and growth conditions Isogenic yeast strains BY4742 (MATa his3D1 leu2D0 lys2D0 ura3D0), Y17334 (MATa his3D1 leu2D0 lys2D0 ura3D0 caf20D::kanMX4) and Y17036 (MATa his3D1 leu2D0 lys2D0 ura3D0 eap1D::kanMX4) were obtained from Euroscarf (Frankfurt, Germany) and grown in synthetic dextrose (SD) minimal medium containing auxo- trophic supplements and grown to A600 = 0.7 for all array experiments. For nitrogen source growth experiments auxotrophic markers were complemented with plasmid encoded gene copies [pRS313-HIS3; pRS314-TRP1; pRS315-LEU2 (16), and pRSK-URA3 LYS2 (a gift from M. Stark, University of Dundee)]. Wild-type or mutant EAP1 plasmids: pJF3896 (HA-EAP1 LEU2 CEN) and pBMK492 (HA-eap1-Y109A,L114A LEU2 CEN) replaced pRS315 where indicated (10). Cells were grown on SD medium containing a single indicated nitrogen source at the following concentrations: 0.1% w/v ammonium sulphate, allantion, 0.02% w/v L-isoleucine, L-leucine, L-tryptophan, L-valine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-serine, 0.01% w/v ammonium chloride, 0.01M urea. All media were adjusted to pH 5.3. Synthetic complete (SC) contained 0.5% w/v ammonium sulphate and 0.2% w/v of a mix of all 20 amino acids, inositol, para-aminobenzoic acid, uracil, adenine as described earlier (17). Agar plates (2% w/v bacto agar) were incubated at 30 C for 2–5 days. Doubling times (Td) in liquid media were determined using 96-well microplate cultures grown in a temperature-controlled absorbance reader with shaking (BMG Labtech). A600 measurements were recorded every 5 min for 20 h. A previously described MS Excel macro was adapted and used to automate Td determinations (18). PUF-TAP tagged strains were a kind gift from Gerber and Herschlag (19). CAF20-Myc and EAP1-Myc tagged versions of each PUF-TAP strain were made using pYM4 and PCR to integrate C-terminal 3Myc-KanMX6 marker as described earlier (20). Similarly, pYM19 (9Myc- HIS3MX6) and pYM13 (TAP-KanMX6) was used to create Myc-tagged and TAP-tagged versions, respectively, of CAF20, EAP1, PUF2, PUF3 and PUF5 in BY4742, Y17334 and Y17036 (20). PCR was used to verify genomic integrations. Polysome fractionation, RNA preparation and microarray analysis Cell extracts, polyribosome fractionation, RNA prepar- ation and array bioinformatics analysis were done exactly as described earlier (21). Complete data sets are