Npl3 Stimulates 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. doi:10.1371/journal.pone.0003273.g002 S411 (S411A, S411E and S411D) were used in the elongation assay to test for this possibility. In addition, a mutant that was previously shown to have a defect in RNA binding (Npl3-120) and a truncated form of Npl3 containing only the two RRMs (aa 121– 280) were also tested [11,12,33]. Elongation stimulation by Npl3- S411D and Npl3-S411E, which may resemble the phosphorylated form of Npl3, was reduced as compared to the wild-type (Figure 2C). In addition, the Npl3-S411A mutant was slightly reduced. This result further highlights the importance of maintaining the integrity of the S411 residue since the S411A substitution differs only in the hydroxyl group present in the serine residue. These results are consistent with the model that S411 phosphorylation can modulate the effect on elongation. Notably, the truncated form of the protein did not stimulate elongation, which reinforces the importance of the RGG/RS domain and S411 in the interaction with RNAP II. Finally, the RRM mutant Npl3-120 also has reduced elongation stimulation, suggesting that RNA binding contributes to this activity. We conclude from these experiments that the positive effect of Npl3 on elongation requires both binding to the nascent RNA and its physical interaction with RNAP II, which may be inhibited by phosphorylation of S411. Npl3 interacts with phosphorylated serine 2 of the CTD The phosphorylated form of serine 2 of the CTD repeat YSPTSPS is associated with elongation [17]. Previous crosslinking experiments demonstrated the presence of Npl3 throughout elongation [9], suggesting a possible link to the CTD and possibly phosphorylated serine 2. An interaction with the CTD was first tested using the RGG/RS peptides. As with purified RNAP II, the unphosphorylated RGG/RS3 was observed to interact with GST- CTD (data not shown). In addition, unphosphorylated or phosphorylated Ser 2, Ser 5 or Ser 2/Ser 5 CTD peptides were used in pull-down experiments with unphosphorylated full-length Npl3 (Figure 2D). The CTD repeat peptide containing the Ser 2 phosphorylation was observed to interact with Npl3, and this result was reproducible. We concluded from these experiments PLoS ONE | www.plosone.org 3 September 2008 | Volume 3 | Issue 9 | e3273