First, upon dissociation of Npl3, the elongation rate of RNAP II would decrease to enhance the window of opportunity for polyA factors to bind to signals in the nascent RNA. It is known that the rate of transcription elongation can affect alternative splicing decisions [48,49], so it is reasonable to assume that a similar relationship can exist between elongation rate and polyadenyla- tion. Interestingly, the elongation promoting activity of Npl3 is weaker in an RNA binding mutant and is inhibited by Rna15, which suggests that binding to the RNA transcript by Npl3 is important for stimulating RNAP II. Binding of Npl3 to the nascent transcript might stabilize interactions between the CTD, RNAP II and the RNA and prevent premature 39 end formation. Accordingly, interactions between the CTD of the mammalian RNAP II with the RNA were shown to be critical to suppress premature termination [50]. Additionally, binding of Npl3 to the RNA may prevent the formation of arrest-inducing secondary structures, or suppress pausing at cryptic polyA sites. A second event upon dissociation of Npl3 would be a shift in the balance of the competition between Npl3 and Rna14/15 for RNA-binding. Several lines of evidence have shown that this competition is important for proper polyadenylation. Transcrip- tion readthrough of weakened polyA signals can be suppressed by mutation/deletion of Npl3 or Cbp20/80, or by overexpression of the polyadenylation factors Rna14 or Hrp1 [11–13]. This competition between Npl3 and Rna15 can be reconstituted in vitro [12], and here we show that this competition can be shifted by the CK2 phosphorylation of Npl3. There are many examples of SR proteins being regulated by phosphorylation. In the case of Srp40, phosphorylation of the RS domain is required for sequence-specific RNA binding [51]. For another RS protein, ASF/SF2, phosphorylation enhances contacts essential for splicing [8,30,52–55]. The identification by mass spectrometry of multiple phosphorylated serines in vivo and in vitro suggests that Npl3 is hyperphosphorylated at the RRM and RGG/RS domains. In the case of Npl3-S411, its phosphorylation decreases crosslinking of the protein to RNA [8](this study), and also functions in autoregulation of the NPL3 transcript [32]. Our study suggests that CK2 is required for the phosphorylation of S411, and we also show that this residue appears to affect Npl3’s interaction with RNAP II. Thus, S411 participates in the binding for both RNA and RNAP II. While this manuscript was in preparation Lund et al reported of an effect of Npl3 in autoregulation of its own transcript [32]. In this study, phosphorylation of Npl3-S411 was observed to increase termination efficiency, thus modulating its own protein levels. Our expression analysis suggests that the termination of NPL3 represents a unique case likely shared by a limited number of genes. Previous work by Steinmetz and co-workers suggested that the choice of termination sites at the NPL3 locus involved a Sen1 anti-termination pathway [56]. In addition, the phosphor- ylation decrease observed for S411 in the cka1 alleles reported in this study did not significantly affect the steady state levels of Npl3, as observed by Lund and co-workers. Therefore, we believe that the autoregulation of Npl3 is likely the result of an activity that is distinct from that mediated by CK2. At this time, the mechanism by which Npl3 functions in its autoregulation by allowing the selection of an alternative termination site remains unresolved. Our study suggests that Npl3 affects elongation and termination in a high percentage of genes (,30%) making this a widely used mechanism in yeast that is likely to function in higher eukaryotes. We predict that metazoan SR-proteins with known functions in hnRNP formation, splicing and mRNA 39end processing might also be able to affect elongation in a manner similar to Npl3. Npl3 Stimulates Elongation Materials and Methods Plasmids and Strains Plasmids pSBEThis7-NPL3 and pSBEThis7-npl3-120 were described previously [11]. For plasmids pSBEThis7-npl3-S411A, - npl3-S411D, and -npl3-S411E, the ORF of NPL3 was amplified by PCR using the oligo: 59-CATGCCATGGCTGAAGCTCAAGA- AACTCACG-39 and 59- CGCGGATCCGCTTACCTGGTTG- GTGCTCTTTCACG-39 for the S411A; or 59- CGCGGATC- CGCTTACCTGGTTGGTTCTCTTTCACG-39 for the S411D; and 59-CGCGGATCCGCTTACCTGGTTGGGTC- TCTTTCACG-39 for the S411E substitution, and cloned into BamHI/NcoI site of pSBEThis7. Plasmid pET21b-CKA1-His6 was described previously [24]. DNA template pAdGR220 used for transcription assays was described previously [28]. Yeast strains used are shown in Table S1. The npl3-S411A strain was kindly provided by M. Lund (UCSF). Protein Purifications E. coli strain Rosetta (DE3) or BL21(DE3) were transformed with pSBEThis7-npl3-S411A, -S411D, -S411E, pET21b-RNA15 or -CKA1-His6 and purified as described previously [11]. Wild-type RNAP II was purified from yeast as described previously [27]. Pull-down and immunoprecipitation assays Npl3 peptides were conjugated to resin using the Carboxylink Immobilization Kit (Pierce). Peptides containing the amino acid sequences: GGYGGYSRGGYGGY (RGG/RS1), RGGYDS- PRGGY (RGG/RS2), YRTRDAPRERSPTR (RGG/RS3), or YRTRDAPRERpSPTR (RGG/RS3p) were synthesized (BCMP/ HMS Biopolymers Lab). Peptides were normalized by OD 280nm. 100 ng of purified RNAP II (A. Ponticelli) was incubated with each of the conjugated peptides overnight at 4uC in IPP-150 buffer (10 mM Tris [pH 7.9], 150 mM NaCl, 1 mM MgOAc, 2 mM CaCl2, 0.1% NP-40, 1 mM DTT with protease inhibitors: 1 mg/ml of aprotinin, leupeptin, antipain and pepstatin-A), and washed using buffer IPP-150 plus 0.01% SDS (sodium dodecyl sulfate) [24]. Amino-terminal biotinylated CTD peptides were previously described [57]. For immunoprecipitations, IgM agarose (Sigma) was conjugated to a His antibody. 100 ng of purified RNAP II was incubated with recombinant His-Npl3, -S411A, -S411D or -S411E for 2 hours at 4uC in IPP-150 buffer and washed as described above. Alternatively, 5 ml of streptavidin- coated magnetic beads (Dynal) were used to bind 1 mg of a CTD peptide repeat (YSPTSPS) containing unphosphorylated, Ser 2, Ser 5 or Ser 2/Ser 5 phosphorylated peptides. 100 ng of recombinant Npl3 was incubated with each CTD peptide and 500 ng Bovine Serum Albumin (BSA), as described previously [57]. For immunoprecipitation of endogenous Npl3 for mass spectrometry analysis, Protein A (Sigma) was conjugated to polyclonal rabbit anti-Npl3 (kindly provided by Lithgow, La Trobe University, Australia) and incubated with 5 mg of whole cell extracts prepared as described previously [58]. All protein was eluted by boiling in the presence of SDS-PAGE loading buffer and resolved using 8% SDS-PAGE. Immunoblotting was done using standard methods using polyclonal mouse anti-CTD (8WG16). Oligo(dC)-tailed template transcription assays Transcription reactions were performed as described previously [28]. Briefly, reactions were carried out at room temperature in the presence of 20 mM HEPES-KOH, pH 7.9, 20 mM Tris-HCl, pH 7.9, 8 mM MgOAc, 100 mM KOAc, 1 mM DTT, 0.5 mg/ml BSA, 3% (vol/vol) glycerol, 8 U of RNasin, 100 ng template DNA and 100 ng RNAP II. Pulse was carried out by the addition of PLoS ONE | www.plosone.org 9 September 2008 | Volume 3 | Issue 9 | e3273