Figure 3.

The rrp40-W195R mutant cells show elevated levels of exosome target transcripts but do not exhibit impaired cytoplasmic exosome function. (A–C) The rrp40-W195R mutant cells show a statistically significant increase in the levels of noncoding RNAs, (A) NEL025c CUT and (B) U4 snRNA, but not (C) ITS-2 rRNA, at 37°. Total RNA from rrp40∆ cells containing only wild-type RRP40 or mutant rrp40 (G8A; W195R) grown at 37° was measured by quantitative RT-PCR using NEL025c CUT, U4 snRNA, and ITS-2 rRNA primers as described in Material and Methods. Relative RNA levels were measured in triplicate biological samples, normalized to a control ALG10 transcript by the ∆∆Ct method, averaged, and shown graphically as fold increase relative to wild-type RRP40. Error bars denote SD. The difference in NEL025c CUT levels between rrp40-W195R and wild-type cells is significant (P = 0.0114; denoted by *) and the difference in U4 snRNA levels between rrp40-W195R and wild-type cells is very significant (P = 0.0090; denoted by **). The differences in RNA levels between rrp40-G8A and wild-type cells are not significant. Statistically significant differences in mean RNA levels were determined using the unpaired t-test. (D) The rrp40 mutant cells do not rescue a his3-nonstop mRNA reporter, which is degraded rapidly by the cytoplasmic RNA exosome (van Hoof et al. 2002), to restore growth on medium lacking histidine, suggesting that rrp40 mutants do not impact cytoplasmic RNA exosome function. As a control, deletion of SKI7, encoding a key cytoplasmic RNA exosome cofactor (van Hoof et al. 2000), rescues the his-nonstop reporter and thus confers growth on medium lacking histidine. The rrp40∆ cells containing wild-type RRP40 or mutant rrp40 (G8A; S87A; W195R; W195A) plasmid and the his3-nonstop mRNA reporter plasmid, and ski7∆ cells containing the his3-nonstop mRNA reporter plasmid were serially diluted and spotted onto minimal medium plates lacking histidine (His⁻) or control plates containing histidine (His⁺) and grown at 37°.