Npl3 Stimulates Elongation Figure 3. Cka1, the alpha catalytic subunit of CK2, phosphorylates Npl3. (A) Recombinant His-Cka1 was incubated with His-Npl3 in the presence of radiolabeled ATP. Phosphorylation of Npl3 is reversed by the addition of increasing concentrations of l-Phosphatase. (B) Using the in vitro kinase assay with mutants of Npl3-S411, this residue was uncovered as an additional phosphorylation site. Wild-type His-Npl3 or S411 point mutants, His-Npl3-S411A, -S411D or -S411E were incubated with His-Cka1, as described for (A) in the presence of radiolabeled-ATP. Coomasie stain representing the concentration of recombinant Npl3 proteins used is shown below. (C) CKA1 mutations show reduced phosphorylation of Npl3. Whole-cell extracts were prepared for cells grown for one hour at the permissive (25uC) or non-permissive (37uC) temperature for wild-type CKA1, cka1-12 or cka1-45, and immunoblot analysis was performed using antibodies specific for phosphorylated or non-phosphorylated Npl3, as indicated. Detection of Rpb3 with specific antibodies is shown as a loading control. doi:10.1371/journal.pone.0003273.g003 be expected to reduce its ability to compete with Rna15. Using a modified UV cross-linking assay with a labeled RNA oligo (N4) thought to be a preferred binding site for Rna15 at GAL7 [7], recombinant Npl3 and Rna15 were tested for binding. Npl3 was observed to compete strongly for binding to the polyA signal (Figure 4A). Titration of Cka1 resulted in an ATP-dependent reduction of Npl3 binding to the RNA, while binding to an oligo corresponding to an Npl3-preferred site was unaffected (N2) (Figure 4B). No cross-linking of Rna15 was detected with the N2 oligo. Rna15 was not phosphorylated by Cka1, nor was binding to the polyA signal affected in reactions with Cka1 lacking Npl3 (data not shown). Thus, we concluded that phosphorylation of Npl3 by Cka1 had a dramatic effect on the RNA binding competition at Rna15-preferred sequences. Competition for RNA binding decreases stimulation of elongation Previous characterization of alleles of NPL3 demonstrated that the function of Npl3 in transcription termination relies on its ability to bind RNA [11,33]. This defect is due to mutations in RRM2, which result in reduced the binding specificity of Npl3 [33,42], and reduce the ability of the protein to compete effectively for binding to polyA/termination sequences [12]. In the elongation assay, the RRM mutant Npl3-120 had slightly reduced elongation stimulation, which suggested that RNA binding was also important for elongation (Figure 2C). We exploited the unspecific RNA binding of Rna15 and its competition with Npl3 to test the RNA binding requirement of Npl3, presuming that binding of Rna15 to the nascent RNA might prevent a stable Npl3/RNA interaction. Stimulation of elongation was therefore measured in the presence of the competing polyA factor Rna15. A two-fold molar excess of Rna15 (relative to Npl3) was added together with Npl3 during the 5-min incubation between the pulse and chase. Figure 4C shows the results for transcription reactions for 0 and 30-minutes after UTP addition. Rna15 alone did not affect the rate of elongation (Figure 4C, lane 8). Npl3 still stimulates elongation in the presence of Rna15, but a decrease in the intensity of the signal for the longer transcripts was seen. Therefore, competition for binding to the RNA transcript between Rna15 and Npl3 may decrease the positive effect on elongation, and strengthens the suggestion that RNA binding plays an important role in the stimulatory effect of Npl3. PLoS ONE | www.plosone.org 5 September 2008 | Volume 3 | Issue 9 | e3273