FIGURE 7.

CAF1 carries the catalytic activity for mRNA deadenylation in S2 cells. (A) Expression of myc-CAF1 or a variant containing a double point mutation in the first exonuclease motif was induced for 20 h in stably transformed S2 cells. Cells were heat-shocked for 30 min at 35.5°C and then allowed to recover at 25°C. RNA was prepared at the indicated times after recovery and digested with RNase H in the presence of an hsp70-specific oligonucleotide. dT indicates Oligo(dT) was included in the RNase H digestion to mark the fully deadenylated RNA. Products were analyzed by Northern blot with probes against the 3′UTR of hsp70 and against U1 RNA serving as loading control. (B) An equivalent experiment was carried out with cells expressing flag tagged CCR4, a CCR4 variant containing a double point mutation in the exo III domain, or a control protein. (C, D) Expression of the tagged proteins was monitored by Western blotting with the antibodies indicated. EndoGI (Temme et al. 2009) served as loading control. (E) Wild-type and mutant CCR4-Flag were precipitated with anti-Flag antibodies and eluted with Flag peptide. Equal amounts of the eluates were separated on a 10% gel, blotted, and probed with antibodies against NOT3 or CCR4.