Npl3 Stimulates Elongation Figure 4. Cka1 disrupts the ability of Npl3 to effectively compete for binding to an Rna15-preferred sequence. (A) Recombinant Npl3, Rna15 and Cka1 were incubated with a radiolabeled RNA oligo (N4), UV cross-linked, and resolved in denaturing 10% SDS-PAGE gels. This RNA oligo consists of an A-rich repeat (described in Materials and Methods), which is the preferred binding site for Rna15 and is commonly found upstream of polyA sites [7]. Representative UV-cross-linking experiments are shown where increasing Cka1 is added to reactions containing Npl3 with Rna15. The graph below each gel shows quantification for the average of three experiments (Npl3, white bars; Rna15, black bars). Binding levels were calculated as fractions relative to a reaction containing the highest concentration of the individual RNA-bound protein (lane 1 for Npl3 and lane 2 for Rna15). Control UV-crosslinking experiment where Cka1 has been added with or without ATP is also shown. Values represent total PhosphorImager units (PIU). (B) Recombinant Npl3, Rna15 and Cka1 were incubated with a radiolabeled U/G/C-rich RNA oligo (N2), UV-cross-linked, and resolved in denaturing 10% SDS-PAGE gels and quantified as described for (A). (C) Similar reactions were performed as described for Figure 1 for 0 and 30 minutes without (lane 1 and 5) or with 78 nM Npl3 (lanes 2–3 for 0 min time-point; and 6–7 for the 30 min time-point). 200 nM Rna15 was added to the transcription reactions in the presence or absence of Npl3 (lanes 3–4 for 0 min time-point; and 7–8 for the 30 min time-point). The oligo(dC)- template is represented next to the gel and the positions of the pause sites at stretches of Ts are indicated. Quantification of the transcription reactions was performed as shown for Figure 2C. doi:10.1371/journal.pone.0003273.g004 Termination defects associated with Npl3 RNA binding and phosphorylation Our previous chromatin immunoprecipitation experiments demonstrated that mutations in the RNA binding domain of Npl3, such as the npl3-120 allele, lead to more efficient termination and increased cross-linking of polyA/termination factors to the 39 end of various genes [11]. Phosphorylation of Npl3 reduces its association with RNA in vivo [10], and its association with an Rna15 preferred binding site in vitro (Figure 4A and [12]. Our results further suggest it is also important for regulating its interaction with RNAP II. Therefore, the expectation was that a mutation that abolishes S411 phosphorylation might result in a termination defect. The S411A mutation was selected for analysis since it more closely resembles an unphosphorylated state, although slight decreases in the elongation and binding assays were detected upon loss of the serine hydroxyl group. The predicted outcome in vivo was that a sustained interaction between unphosphorylated Npl3, the polymerase and RNA, would result in failure of RNAP II to release and terminate, and a more effective competition with Rna15 for recognition of termination/polyA signals, based on the assumption that both mechanisms are coupled in vivo. This prediction was tested by RNA expression analysis using whole genome tiled arrays consisting of 25-mer reverse probes with 5 base pair spacing. RNA was extracted from NPL3, npl3-120 and npl3-S411A strains, followed by synthesis of labeled sense-strand cDNA and hybridization to the tiled arrays [43]. The intensities of two independent experiments were used to plot the ratio of mutant to wild-type. Shown in Figure 5A–B are plots for three representative genes (FBA1, MPE1 and TDH3). The ratio of mutant to wild-type expression levels showed a clear difference at the 39 ends. For the npl3-120 strain, a decrease in transcript levels near the 39end of the genes was observed (Figure 5 and Figures S2, S3 and S4), consistent with previous experiments showing that this mutation PLoS ONE | www.plosone.org 6 September 2008 | Volume 3 | Issue 9 | e3273