The rrp4-G226D variant cells show elevated levels of some but not all RNA exosome target transcripts. (A) The rrp4-G226D cells exhibit greater accumulation of 7S pre-RNA compared to RRP4 and rrp40-W195R cells grown at 37°C. Total RNA from RRP40, rrp40-G8A, rrp40-W195R, RRP4, and rrp4-G226D cells grown at 37°C was analyzed by northern blotting with a 5.8S-ITS2 probe to detect 7S pre-rRNA. Mature 5.8S rRNA and 5S rRNA were detected by methylene blue staining as a loading control. The 7S pre-rRNA is normally processed to mature 5.8S rRNA by 3′–5′ decay of the internal transcribed spacer 2 (ITS2) via the nuclear RNA exosome (Mitchell et al. 1996; Allmang et al. 1999). All lanes are imaged from the same northern blot with a gap in the loading indicated by the white line. The simplified schematics to the right illustrate the processing steps of 7S rRNA precursor following endonucleolytic cleavage from the larger 27S precursor (indicated by white triangles). (B) The rrp4-G226D cells show an elevated steady-state level of 3′-extended pre-U4 snRNA relative to RRP4 cells at 37°C. (C) The rrp4-G226D cells exhibit an increased steady-state level of U14 (snR128) snoRNA but not snR44 snoRNA relative to RRP4 cells at 37°C. (D) The rrp4-G226D cells show an elevated steady-state level of mature and extended precursor TLC1 telomerase component ncRNA relative to RRP4 cells at 37°C. In B–D, total RNA was isolated from cells grown at 37°C and transcript levels were measured by RT-qPCR using gene specific primers, normalized relative to RRP4, and graphed as described in Materials and Methods. Gene specific primer sequences are summarized in Supplemental Table S2 and their locations within the transcript are graphically represented by the cartoons above each bar graph. Within the cartoon transcript, the box represents the body of the mature transcript. Error bars represent standard error of the mean from three biological replicates. Statistical significance of the RNA levels in rrp4-G226D cells relative to RRP4 cells is denoted by an asterisk (*P-value ≤0.05; **P-value ≤0.01).
