hsRNAs expressed in the absence of nuclear PABPs still direct protein synthesis. (A) [35S]-Met protein labeling analysis of the indicated cell cultures (SDS-PAGE; bottom) following the experimental scheme displayed at the top. Rapamycin, or DMSO (−rapamycin), was added 20 min before heat induction, followed by [35S]-Met labeling for one of three indicated time periods (gray horizontal arrows): (1) at 25°C for 5–15 min before the temperature shift (−15′–5′), or (2) for 5–15 min (5′–15′) or (3) for 15–25 min (15′–25′) after the 38°C shift. Markers for heat shock proteins are indicated on the right. (B) Quantification of Hsp104p expression from A. Incorporation of [35S]-Met into Hsp104p in each gel lane was normalized to the total [35S]-Met signal from proteins <50 kDa (see Supplemental Fig. S5A). Bars display averaged values normalized to the “MEX67-AA, − rapamycin, 38°C 5′–15′” samples. Individual values from two independent replicates are shown as dots. (C) RNase H/Northern blotting analysis of HSP104 mRNA 3′ ends as in Figure 1B, but in conditions used for [35S]-Met protein labeling. RNA samples were from cells harvested 15 min after transfer to 38°C. Additional time points are shown in Supplemental Figure S5C. Note the loss of HSP104 RNA signal and the appearance of hyperadenylated transcripts following nuclear depletions of Mex67p and Nab2p.
