FIG. 7.
yra1, sub2, and Δhpr1 mutants induce defects in mRNA accumulation. (A) Northern blot analysis of PMA1 mRNAs in wild-type or GFP-yra1-8, sub2-201, and Δhpr1 mutant strains grown at 25°C (lanes 1 to 4) or shifted to 37°C for 45 min (lanes 5 to 8). The PMA1 signals were normalized to 18S rRNA and expressed as a percentage of that of the wild type, as indicated (% wt). (B) Wild-type or GFP-yra1-8 (FSY1568), sub2-201 (FSY1613), and Δhpr1 (FSY1624) mutant strains, transformed with plasmids (2μm URA3) containing the β-galactosidase or YAT1 genes driven by a galactose-inducible promoter, were grown to mid-log phase in selective medium containing 2% lactate-2% glycerol-0.05% glucose and induced with 3% galactose-1% raffinose for 150 min at 25 or 37°C, as shown above the lanes. Total RNA was analyzed by primer extension with 32P-labeled oligonucleotides specific for β-galactosidase, YAT1, or the endogenous GAL1 gene transcripts, as indicated. A primer specific for U1 snRNA was added to the reactions as an internal control for loading. The primer-extended bands were quantified with an Instant Imager apparatus and normalized to U1 snRNA. The transcript levels in mutant strains were expressed as a percentage of that of the wild type, as indicated at the bottom of each gel (% wt). (C) The YRA1 (FSY1026) or YRA1 Δrrp6 (FSY1621) strain was shuffled, as described for Fig. 6B, with plasmid Lac111-YRA1 Gen (pFS2525; lanes 1 and 2) or Lac111-yra1-8 (pFS2328; lanes 3 and 4) and transformed with the galactose-inducible β-galactosidase reporter construct (pLGSD5). Transformants were grown, and RNAs were analyzed as for panel B, except that the galactose induction was performed at 30°C.