Validation of the differentially expressed transcripts identified in the RNA-seq confirms that the levels of key mRNAs and CUTs are significantly altered in rrp4-G226D cells and reveals that some of these transcripts are not changed in rrp40-W195R cells. (A) The steady-state levels of noncoding, cryptic unstable transcripts, CUT501, CUT770, and CUT896, are significantly increased in rrp4-G226D cells compared to control. The CUT770 level is also increased but the CUT501 and CUT896 levels are not altered in rrp40-W195R cells. (B) The steady-state levels of ribosomal protein gene mRNAs, RPS3 and RPL15A, and inositol-3-phosphate synthase mRNA, INO1, are significantly decreased in rrp4-G226D and rrp40-W195R cells relative to control RRP4/40 cells. (C) The steady-state level of peptidyl tRNA hydrolase 4 mRNA, PTH4, is significantly increased in rrp4-G226D and rrp40-W195R cells relative to controls, whereas the levels of hexokinase isoenzyme 2 mRNA, HXK2, and glyceraldehyde-3-phosphate dehydrogenase isozyme 1 mRNA, TDH1, are significantly decreased in rrp4-G226D compared to control. The HXK2 and TDH1 levels are not altered in rrp40-W195R cells. (D) The steady-state levels of RNA exosome/termination cofactor mRNAs, NRD1, and NAB3, are significantly increased in rrp4-G226D cells compared to controls. The NRD1 level is increased, but the NAB3 level is not altered in rrp40-W195R cells. In A–D, total RNA was isolated from cells grown at 37°C and transcript levels were measured by RT-qPCR using gene specific primers (Supplemental Table S2), normalized relative to RRP4/40, and graphed as described in Materials and Methods. Error bars represent standard error of the mean from three biological replicates. Statistical significance of the RNA levels in rrp4-G226D and rrp40-W195R cells relative to control RRP4/40 cells or between rrp4/40 mutants is denoted by asterisks (*P-value ≤0.05; **P-value ≤0.01).
