for the regulation of mammalian promoter-dependent elements (DPE), and was shown to have an anti-termination effect [21–23]. CK2 also phosphorylates factors within the polyA/termination machinery that are important for 39 end processing [24,25]. Here we show that Npl3 directly interacts with phosphorylated serine 2 of the CTD and stimulates the elongation rate of RNAP II in vitro. Association with polymerase is inhibited by phosphorylation of Npl3 on S411. Stimulation of elongation is also reduced by a mutation in the Npl3 RNA binding domain, indicating that RNA binding is important. CK2 is shown to be required for Npl3’s phosphorylation, and alter the competition between Npl3 and Rna15 for binding to the RNA. These results demonstrate that Npl3 functions as both a positive transcription elongation factor and an anti-terminator and that these activities are regulated by phosphorylation. Results Npl3 affects the rate of transcription elongation Mutations in or near the second RNA Recognition Motif (RRM) of NPL3 have been shown to enhance termination [11]. These same npl3 alleles confer sensitivity to MPA (mycophenolic acid) and 6AU (6-azauracil), phenotypes suggestive of elongation defects. To test for an effect of Npl3 on the rate of RNAP II elongation, an in vitro transcription assay containing purified RNAP II and a promoterless dC-tailed template was used [26–28]. In this assay, the polymerase binds to the single-stranded oligo(dC)-tail and initiates transcription in the presence of ATP/GTP/[a-32P]CTP [26]. In the absence of UTP, the polymerase is forced to pause at the first non-template T stretch (135 nt = T3) (Figure 1, lanes 1 and 2). After a 30-minute labeling incubation, the reaction is split and incubated for an additional 5-minutes with buffer or Npl3. The labeled transcripts are then extended with limiting UTP and excess unlabeled CTP for 2 to 60 minutes in the absence (Figure 1, odd lanes), or presence of Npl3 (Figure 1, even lanes). Limiting the chase lowers the rate of nucleotide addition through dTTP regions on the template, causing the accumulation of transcripts that are ,250 nt in length. These products are eventually chased into longer products. A notable increase in the rate of elongation by RNAP II was observed in the presence of Npl3 (Figure 1). These results suggest that Npl3 functions as a positive elongation factor in the absence of termination factors. Unphosphorylated Npl3 interacts with RNAP II and mediates the elongation effect The C-terminal RS domain of Npl3 contains three serine/ proline [29] and six serine/arginine (SR) repeats that are interspersed within a region rich in arginines and glycines (RGG/RS domain). RS domains in other proteins can mediate protein-protein interactions [30]. Phosphorylation of RS domains also functions in the recruitment of these proteins to sites of transcription (e.g., SF2/ASF) [29]. In addition, RS domain phosphorylation can affect RNA binding affinity [8,30,31]. Npl3 and RNAP II co-immunoprecipitate from yeast extracts, although it is unclear if this interaction is direct or indirect [9]. In order to test the role of the RGG/RS domain of Npl3 plus its phosphorylation in this interaction, peptides corresponding to three repeats within the RGG/RS domain were conjugated to a resin and used in pull-down experiments with purified RNAP II (Figure 2A). Peptide RGG/RS1 corresponds to the SR repeat (GGYGGYSRGGYGGY), peptide RGG/RS2 corresponds to the SP repeat (RGGYDSPRGGY), and peptide RGG/RS3 corre- sponds to the SP repeat containing serine 411 (YRTRDA- PRERSPTR). Since S411 is phosphorylated in vivo, and it has been extensively characterized [10,32], we also included a phosphor- Npl3 Stimulates Elongation Figure 1. Npl3 stimulates RNAP II elongation. Oligo(dC)-tail templates were used in transcription reactions with or without 78 nM Npl3, as indicated. Reactions were pulsed by the addition of ATP, GTP, and a32P-labeled CTP for 30 minutes as described in Materials & Methods. Transcripts were chased by the addition of excess CTP and limiting UTP for the indicated times. A schematic of the experimental scheme is shown on top. The oligo(dC)-template is represented next to the gel and the positions of the pause sites at stretches of Ts are indicated. doi:10.1371/journal.pone.0003273.g001 ylated form of this peptide (YRTRDAPRERpSPTR). No RNAP II was detected as interacting with the RGG/RS1 or RGG/RS2 peptides (Figure 2A). However, significant binding was observed with the unphosphorylated RGG/RS3 peptide. Binding to the phosphorylated RGG/RS3 was much weaker. These results suggest that Npl3 interacts directly with RNAP II primarily through RGG/RS3 in its unphosphorylated form. The contribution of S411 in RGG/RS3 to the interaction between Npl3 and RNAP II was further tested by immunopre- cipitation with full-length Npl3 and three different mutants of S411 (S411A, S411E and S411D), which resemble unpho- sphorylated or phosphorylated forms of the protein. To rule out possible conformational changes due to the mutations, the point mutants were tested for RNA binding and found to retain their ability to bind RNA (data not shown). Purified RNAP II was incubated with recombinant histidine-tagged Npl3, Npl3-S411A, - S411D or -S411E, and reactions were immunoprecipitated using anti-His antibody and immunoblotted for RNAP II. Wild-type Npl3 bound to RNAP II (Figure 2B), while mutants Npl3- S411A, -S411D and -S411E showed further decrease. This result suggests that changes to S411 affect the interaction, particularly those mutations that resemble a phosphorylated state. The interaction between unphosphorylated RGG/RS3-S411 and RNAP II suggested that Npl3’s effect on elongation might be regulated through phosphorylation of S411. The three mutants of PLoS ONE | www.plosone.org 2 September 2008 | Volume 3 | Issue 9 | e3273