Figure 1

Influence of putative cofactors on Pa-exosome poly-A RNA binding. (A) Electrophoretic mobility shift assays with radiolabeled 14-mer poly-rA probe incubated with fixed amounts of the indicated purified proteins (100 pmol of either Pa1135 or PaNip7, or 200 pmol of PaSBDS), and increasing amounts of the exosome complexes (1, 10, or 20 pmol of either RNase PH ring, PaRrp4-exosome, or PaCsl4-exosome). Lanes 13-15, 20 pmol of exosome complexes. Proteins were incubated with 1 pmol RNA at 37°C for 30 min, and 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 and protein-bound RNAs are indicated on the right-hand side. (B-D) Quantitation of bands visualized in RNA binding assay. The ratio of protein-bound RNA over free 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, 200 pmol PaSBDS, or 100 pmol Pa1135. (B) Effect of the three tested proteins on RNase PH ring exosome complex. (C) Effect of the proteins on the PaRrp4-exosome. (D) Effect on the PaCsl4-exosome.