Identifying eIF4E-binding protein translationally-controlled transcripts reveals links to mRNAs bound by specific PUF proteins

Abstract

eIF4E-binding proteins (4E-BPs) regulate translation of mRNAs in eukaryotes. However the extent to which specific mRNA targets are regulated by 4E-BPs remains unknown. We performed translational profiling by microarray analysis of polysome and monosome associated mRNAs in wild-type and mutant cells to identify mRNAs in yeast regulated by the 4E-BPs Caf20p and Eap1p; the first-global comparison of 4E-BP target mRNAs. We find that yeast 4E-BPs modulate the translation of >1000 genes. Most target mRNAs differ between the 4E-BPs revealing mRNA specificity for translational control by each 4E-BP. This is supported by observations that eap1Δ and caf20Δ cells have different nitrogen source utilization defects, implying different mRNA targets. To account for the mRNA specificity shown by each 4E-BP, we found correlations between our data sets and previously determined targets of yeast mRNA-binding proteins. We used affinity chromatography experiments to uncover specific RNA-stabilized complexes formed between Caf20p and Puf4p/Puf5p and between Eap1p and Puf1p/Puf2p. Thus the combined action of each 4E-BP with specific 3'-UTR-binding proteins mediates mRNA-specific translational control in yeast, showing that this form of translational control is more widely employed than previously thought.

Figure 1.

Widespread translational changes are associated with each 4E-BP deletion. (A) A₂₅₄ traces showing polysome profiles from exponential phase cultures for the strains used. RNA fractions collected were pooled into monosomal (M) or polysomal (P) fractions from wild-type (w) caf20Δ (c) and eap1Δ (e) cells for translational-profiling array experiments. (downward arrow) “halfmer” peaks. (B) Graphical representation of global changes in translation accompanying deletion of CAF20 or (C) EAP1. Mean polysome-to-monosome ratio intensities for each probeset from mutant (y-axis) is plotted against the wild-type (x-axis). Points above or below a log₂ = 0.9 cut-off are considered significant changes (coloured red and blue, respectively). Plots (D) and (E) as B and C except spots representing translationally regulated genes (red and blue) from the eap1Δ plot (C) are shown on the caf20Δ plot (D), and regulated genes from the caf20Δ plot (B) on the eap1Δ plot (E). (F) Venn-style diagram showing the number of genes altered (in parenthesis) and the overlap between data sets. Numbers in black filled circles represent ‘potentiated’ genes (see text). (G) Immunoblots of Caf20p-Myc and Eap1-Myc levels in strains indicated. eIF2γ (GCD11) loading control from Caf20-Myc tagged cells is also shown.

Figure 2.

qPCR and immunoblotting confirms translational profiling. (A) Quantitative reverse-transcription PCR analysis of translation state for qPCR (light grey bars ±SD, n = 3) and array data (black bars) for indicated transcripts (plotted on a log₂ scale so that reduced polysome association in mutant cells is shown as a negative number). (B) Top: total protein levels of Sec9p, Taf7p, Taf3p, Cic1p, Lsm8p, Pub1p and Ade2p were analysed by immmunoblot analysis in indicated yeast strains. Experiments were done in triplicate. Loading control Arp2p (Actin Related Protein 2) is shown beneath each. Middle: densitometry analysis for each protein relative to Arp2p is beneath each panel (dark grey bars). Bottom: polysome/monosome [P/M] ratios from translational profiling (light grey bars).

Figure 3.

Functional classification of regulated mRNAs. Yeast ‘GO Slim’ summary of significantly enriched and under-represented gene Ontology classes calculated using the hypergeometric distribution (performed at www.yeastgenome.org), see Supplementary Table S10 for more details including all calculated P-values. Bold text indicates most over-represented classes (P < 0.00001) in one or more experiment.

Figure 4.

eap1Δ and caf20Δ 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 µl) onto the indicated media. Growth was scored on a scale of 0 (none), +/− (minimal) to 3+ (wild-type).

Figure 5.

Translation state change for Puf1-5p associated mRNAs in caf20Δ and eap1Δ 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 caf20Δ and eap1Δ, 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.

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 (αTAP) and lower with anti-Myc antibodies. I, input [10 µg total protein, P = Pellet (0.5mg) S = supernatant (10 µg unbound fraction)]. The immunoblots are representative of three independent experiments.