Figure 4.

ER stress-induced mRNA splicing replaces the C-terminal portion of Hac1p. (A) Structures of the 1.4-kb and 1.2-kb HAC1 mRNAs are schematically presented. Locations of primers 1–6 for RT-PCR and DNA probes A-E for Northern blot hybridization are also indicated. The 1.4-kb mRNA can encode a bZIP protein of 230 aa, whereas the 1.2-kb mRNA resulting from ER-stress–induced splicing can encode a bZIP protein of 238 aa. Shown below are the nucleotide sequences around the 5′ and 3′ splice sites. The splicing deletes the internal 252 nt from nt 662 to 913 (the A of the first ATG codon is set as +1), resulting in the replacement of the C-terminal 10 aa with a stretch of 18 aa encoded by the second exon. (B) Total RNAs were extracted from the ERN⁺ strain grown for 1 h in the presence (+) or absence (−) of tunicamycin (TM). HAC1 cDNA was prepared by RT-PCR using a fixed 5′ primer 1 and various 3′ primers (primers 2–6), and the products were analyzed by 1% agarose gel electrophoresis. The amounts of PCR products did not reflect the amounts of mRNA well due to excessive cycles used. Positions of the 100-bp ladder DNA size markers are indicated. (C) Total RNAs prepared as in B were analyzed by Northern blot hybridization using the various DNA probes indicated in A, which had been labeled with [³²P]dCTP by PCR. Positions of the 1.4-kb and 1.2-kb HAC1 mRNAs are indicated.