As S1 nuclease protection assays were performed using total RNA isolated from cells submitted to a higher concentration of cadmium (250 μM) than those used in the construction of the stress libraries (50 and 100 μM), we also

performed these assays with RNA isolated from cells submitted to 25, 50 and 100 μM CdCl2 to verify the effect of different cadmium concentrations on hsp70-1 intron splicing. We observed a more pronounced block in the processing of hsp70-1 intron when B. emersonii cells were treated with 100 μM CdCl2 than with 50 μM CdCl2, while with 25 μM CdCl2 no inhibition of splicing was Cell Cycle inhibitor detected (Additional file 3). These results indicate that

inhibition of splicing by cadmium treatment can be dose-dependent, consistent with our observation that a larger number MK-0457 order of iESTs is found in the cDNA library check details constructed from cells submitted to 100 μM CdCl2 (CDC) than from cells exposed to 50 μM CdCl2 (CDM) (Additional file 1). Induction of thermotolerance by incubation at moderate temperatures restores splicing To test whether a pretreatment at moderate heat shock temperatures could exert some effect on mRNA processing in B. emersonii cells, S1 nuclease protection assays were performed using RNA samples from cells incubated at 38°C for 30 min prior to exposure to extreme heat shock temperature (42°C) or cadmium treatment. In these experiments, it was possible to observe

that splicing inhibition occurring at 42°C could be completely reversed if pre-incubation at 38°C was associated with incubation at 27°C for 30 min after exposure to the extreme heat shock temperature (Figure 4A), which could be considered a recovery period. Furthermore, protein Thymidylate synthase synthesis was necessary during the entire experiment, as addition of cycloheximide (10 μg/ml) at any time during cell incubation at the various temperatures prevented complete recovery of the cells’ capacity to carry out splicing of hsp70-1 intron (not shown). In particular, addition of cycloheximide before the pre-incubation step at 38°C, revealed that this treatment is essential for reversing splicing inhibition, as no spliced mRNA is detected under this condition (not shown). In the case of splicing inhibition due to exposure to cadmium, pre-incubation at 38°C prior to heavy metal treatment was also capable of reversing inhibition (Figure 4B), but complete recovery of the splicing capacity was observed only if exposure to cadmium was followed by incubation at 27°C in the absence of the metal (Figure 4B).