Figure 8

Caf20p self-regulates its own transcript. (A) Transcripts overrepresented in the eIF4E pull-downs relative to either eIF4G1 (light blue) or eIF4G2 (pink). The data are taken from the GLM model presented in Figure 3B. (B) A three-dimensional surface plot of P-values detailing the level of significance for the enrichment of the individual closed loop/eIF4E-BP transcripts across the six RIP-seq experiments. (C) A semi-quantitative RT-PCR validation of Caf20p protein’s association with its own transcript using primers designed to the regions 1 to 6 depicted in the figure (detailed in Additional file 6). The level from these regions of the CAF20 transcript was determined in TAP affinity purified samples from the CAF20-TAP strains relative to wild-type (WT) strains. (D) A diagram depicting two possible models by which Caf20p could interact with its own transcript to regulate protein production. (E) TAP affinity purification and western blot analysis from eIF4E-TAP tagged strains, investigating the association of eIF4E with both endogenous Caf20p protein and Flag-tagged wild-type Caf20p or Flag-tagged Caf20^m2p (which has had the eIF4E binding region mutated). (F) Validation of the specificity of RT-PCR primers using total RNA from the strains depicted under the bar chart for either endogenous CAF20 transcripts or Flag-tagged CAF20 transcripts (Additional file 6). Error bars are ± standard error from three replicate experiments. (G) qRT-PCR for the endogenous and Flag-tagged CAF20 transcripts from an eIF4E-TAP affinity purification using the primers validated above. The CAF20 or CAF20-fl transcripts are quantified in the IP samples relative to total RNA for the strains listed. Error bars are ± standard error from three replicate experiments. (H) Western blot analysis using extracts from caf20 deletion strains transformed with either centromeric (low copy) plasmids bearing either wild-type CAF20-fl gene or the m2 mutant of CAF20-fl. Three different single transformants are analyzed for each strain and the blots are probed with anti-Flag antibodies to detect Caf20-fl relative to control anti-eIF4A antibodies.