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. 2024 Jul 14;300(8):107571. doi: 10.1016/j.jbc.2024.107571

Figure 4.

Figure 4

Increasing the level of the Rrp41-L187P protein improves RNA exosome function but not to the same level as WT Rrp41. Myc-tagged Rrp41 or Rrp41-L187P (A and B) or untagged Rrp41 or Rrp41-L187P (C–E) was expressed from either a low copy (CEN) or high copy (2μ) plasmid in rrp41Δ cells as the sole source of Rrp41 in the cells. A, as assessed by immunoblotting with an anti-Myc antibody, steady-state levels of both Rrp41-Myc and Rrp41-L187P-Myc are significantly increased from the high copy (2μ) plasmid compared to the low copy (CEN) plasmid. Samples were probed with anti-Pgk1 antibody to detect Pgk1 as a loading control. The stain-free signal on the immunoblot is also shown as a loading control. B, to quantitate the fold-change in levels of Rrp41 and Rrp41-L187P from high copy versus low copy plasmids, the level of high copy (2μ) Rrp41 or Rrp41-L187P was normalized to low copy (CEN) Rrp41 or Rrp41-L187P, respectively. Rrp41 protein expressed from the low copy (CEN) plasmid was therefore set to 1.0. As shown in the bar graphs, for results at 30 °C (top) and 37 °C (bottom), the levels of Rrp41 are increased 16.3-fold while levels of Rrp41-L187P are increased 14.3-fold for the high copy (2μ) plasmid versus the low copy (CEN) plasmid at 30 °C. Levels of Rrp41 are increased 6.5-fold while levels of Rrp41-L187P are increased 25.8-fold from the high copy (2μ) plasmid versus the low copy (CEN) plasmid at 37 °C. Error bars represent SEM. Statistical significance is calculated by a Student’s t test (∗p-value ≤ 0.05). C, the rrp41Δ cells expressing Rrp41-L187P from the high copy (2μ) plasmid show improved growth compared to cells expressing Rrp41-L187P from the low copy (CEN) plasmid at 25 °C and 30 °C. The rrp41Δ cells containing RRP41 or rrp41-L187P low copy or high copy plasmid were serially diluted, spotted, and grown at the indicated temperatures for 2 to 3 days. D and E, the steady-state levels of RNA exosome target transcripts (D) U4 pre-snRNA and (E) TLC1 telomerase component pre-ncRNA exhibit a statistically significant decrease in rrp41Δ cells expressing Rrp41-L187P from the high copy (2μ) plasmid compared to cells expressing Rrp41-L187P from the low copy (CEN) plasmid. Total RNA was isolated from rrp41Δ cells expressing either low copy (CEN) or high copy (2μ) RRP41 or rrp41-L187P plasmid grown at 30 °C, and transcript levels were measured by RT-qPCR using gene-specific primers (illustrated above each graph), normalized relative to the low copy (CEN) RRP41 sample, which was set to 1.0. Error bars represent SEM from three biological replicates. Statistical significance was calculated by a Student’s t test (∗∗p-value ≤ 0.01; ∗∗∗p-value ≤ 0.001).