Unphosphorylated SR-Like Protein Npl3 Stimulates RNA Polymerase II Elongation
Figure 2
Npl3 interaction with RNAP II is affected by S411 phosphorylation.
(A) Unphosphorylated peptide RGG/RS1 (lane 1), RGG/RS2 (lane 2), RGG/RS3 (lane 3) and phosphorylated RGG/RS3 (lane 4) were used in pull-down assays with purified RNAP II, as indicated, and one fifth of the input is shown in lane 5. The precipitated sample (P) and supernatant (S) were analyzed by immunoblotting. (B) Full-length Npl3 or S411 mutants were used in immunoprecipitation assays with purified RNAP II (100 ng), as indicated. A control lane with RNAP II (10 ng) is shown in lane 13. (C) Oligo(dC)-tail transcription reactions were performed as described in Figure 1 for 30 minutes with RNAP II only (lane 1) or with equivalent concentrations of Npl3 (lane 2); Npl3-S411A (lane 3); -S411D (lane 4); -S411E (lane 5); Npl3-truncated (RRMs only, aa 121–280) (lane 6); or Npl3-120 (lane 7). Quantification of the transcription reactions was performed by calculating the ratio of the 450 nt to the 250 nt bands and normalized using the RNAP II only lane, as shown in the graph. The experiment was done twice, and a representative gel was chosen. (D) Unphosphorylated (lane 1 and 6), or phosphorylated Ser 5 (lane 2 and 7), Ser 2 (lane 3 and 8), and Ser 2/Ser 5 (lane 4 and 9) CTD peptides were used in pull-down assays with full-length Npl3, as indicated, and input is shown in lane 11. The IP and S shown were analyzed as described for (C) using antibodies specific for Npl3. Control Npl3 with no peptide is shown in lanes 5 and 10.