Figure 3

Influence of PaNip7 on Pa-exosome RNA binding and degradation of poly-AU RNA. (A) RNase protection assay with 1 pmol radiolabeled 21-mer poly-rAU probe incubated with varying amounts of the exosome complexes (1, 10, or 20 pmol of either RNase PH ring, PaRrp4-exosome, or PaCsl4-exosome), in the absence or presence of 100 pmol PaNip7. RNA was incubated with either PaNip7 or buffer at 37°C for 30 min, followed by the addition of the exosome complexes and further incubation at 65°C for 15 min. RNA-protein complexes were fractionated on 8% native polyacrylamide gels and visualized by phosphorimaging. -, No protein added to the reaction. Bands corresponding to free RNA oligo, protein-RNA complexes, and RNA degradation products are indicated on the right-hand side. (B-D) Quantitation of bands visualized on native polyacrylamide gels after RNase protection assay. The ratio of degradation product over total RNA in each lane was calculated for the three concentrations of exosome complexes used (1, 10, or 20 pmol), in absence or presence of 100 pmol PaNip7. (B) Effect of PaNip7 on the RNA degradation by the RNase PH ring exosome complex. (C) Effect of PaNip7 on the PaRrp4-exosome. (D) Effect of PaNip7 on the PaCsl4-exosome.