bZIP60 mRNA splicing is stimulated by chemicals that trigger the UPR.
A, Schematic representation of two approaches used to detect the bZIP60 mRNA spliced forms. Primers sets flanking the putative splicing regions (solid arrows) are indicated (Top) to amplify bZIP60u and bZIP60s forms using RT-PCR. Alternative, RT-PCR products are subsequently digested using Alw21I restriction enzyme (Bottom). The latter approach will highlight the length differences between bZIP60u and bZIP60s since the Alw21I restriction site is present in bZIP60u and absent in bZIP60s. bZIP60u and bZIP60s PCR products upon digestion are shown. B, Processing of bZIP60 mRNA was analyzed by gel electrophoresis in agarose (3.5% p/v). RT-PCR products (Top) or RT-PCR products digested with Alw21I (bottom) were obtained from RNA samples of Arabidopsis seedlings (6-day-old) treated for 2 hours with several chemicals that trigger the UPR (Tm 5 µg/mL; DTT 5 mM; CPA 100 µg/mL; Thapsigargin 500 nM). DMSO and water-treated samples served as mock controls for chemicals. Asterisk indicates a hybrid band formed by the bZIP60u and bZIP60s PCR products. Such hybrid band has been also observed and documented in RT-PCR analysis of XBP-1 processing . Elongation factor 1 alpha (EF1α) expression served as a control. All the experiments were performed at least three times with similar results.