Fig. 7. Ubiquitination is required for the recovery of nucleocytoplasmic transport and translation following heat shock.

(A) Immunoblot showing formation and dissolution of poly-ubiquitinated protein-mRNA complex during heat shock and isolated by oligo(dT) resin. Where indicated, TAK243 was added to media 30 min prior to heat shock and maintained for the duration of the experiment. In drug-treated unstressed samples, HEK293T cells were incubated with TAK243 for 180 min prior to lysis. (B) HEK293T cells expressing nucleocytoplasmic transport reporter NLS-tdTomato-NES were fixed and imaged after no stress, 60 min heat shock, or 60 min heat shock and 120 min recovery. Cells were treated with TAK243 or DMSO for 30 min prior to heat shock or for a total of 120 min in non-heat-shocked cells. Nuclear and cytoplasmic boundaries are indicated with dashed yellow and white lines respectively. (C) Quantification of nucleocytoplasmic ratio of tdTomato intensity from cell images described in (B) from 20–30 cells for each condition. Error bars indicate s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ANOVA with Tukey’s test. (D) Immunoblotting of HEK293T cells treated with puromycin to label nascent transcripts for 30 min prior to heat shock and recovery in the presence or absence of TAK243. Cells were lysed at indicated times and translational activity was analyzed by immunoblotting for puromycin. (E) Quantification of immunoblots shown in (D). Average and individual values for puromycin signal are shown for two replicate experiments. n.s., not significant, *P < 0.05, **P < 0.01, student’s t-test. (F-G) Proposed model illustrating heat shock-induced ubiquitination associated with (F) mRNP remodeling and (G) stress granule formation and shutdown of cellular activities, and the requirement for active ubiquitination for reversal of these processes during recovery.