co-transcriptional recruitment of eukaryotic RNA binding proteins is now well established, to our knowledge a direct stimulation of elongation by SR or hnRNP proteins has not been previously reported. No such stimulation is seen with the RNA binding protein Rna15 or the truncated Npl3 RRM domain, arguing that the Npl3 effect is not due to non-specific interaction with RNA. The data presented is consistent with a model where Npl3 functions in two ways: it directly interacts with phosphorylated serine 2 of the polymerase CTD to promote elongation and at the same time binds RNA to antagonize binding of the polyA/ termination factor Rna15 (Figure 6) [9,11]. This combination of activities resembles the l-related phage 82 anti-terminator Q82 that, together with the NusA protein, decreases pausing of the bacterial RNA polymerase and protects the RNA transcript from termination factors [47]. Phosphorylation of Npl3 reduces its interaction with RNAP II and RNA. It is interesting to speculate that the targeted phosphorylation of Npl3 at 39 ends of genes might help trigger termination. This model is supported by chromatin immunoprecipitation experiments where crosslinking of Npl3 was observed to decrease downstream of the polyA site [11], by the expression analysis presented here where mutations at S411 or in the second RRM of Npl3 were shown to Npl3 Stimulates Elongation specifically affect transcript signals at 39 ends of genes, and by UV- crosslinking where the Cka1 phosphorylation decreased Npl3’s competition for binding to polyA signals. In addition, a recent report by Lund et al suggests that phosphorylation is involved the autoregulation of the NPL3 transcript [32]. The cytosolic kinase Sky1 phosphorylates Npl3 [8]. Thus far, no other kinase had been linked to the phosphorylation of Npl3. CK2 is the only kinase reported to phosphorylate proteins involved in mRNA 39 end formation. Phosphorylation of 39 end processing factors Pta1 and Fip1 by CK2 affects their ability to cleave and polyadenylate the mRNA [24]. Therefore, the phosphorylation of Npl3 by CK2 shown here would support a targeted phosphorylation at the 39 end. The existence of nuclear and cytosolic kinases that phosphorylate an mRNA shuttling protein at an identical residue suggests that the same phosphorylation switch, which functions by modulating protein-protein and -RNA interactions can be activated in different compartments of the cell. It remains to be determined what other kinases operate at the 39 end or whether additional transcription- associated kinases can control the phosphorylation state of Npl3 to regulate its activity at sites of transcription. The model postulates several events that would affect assembly of the polyA/termination machinery at the 39 end (Figure 6). Figure 6. Two activities of Npl3 promote elongation. Npl3 directly stimulates the elongation rate of RNAP II by physically interacting with phospho-Ser 2 of the CTD and the RNA. Phosphorylation regulates Npl3’s interaction with RNAP II and RNA, and promotes binding of polyadenylation/termination factors. Binding of Npl3 to the nascent RNA may stabilize interactions between CTD-RNAP II and the RNA. Npl3, white circle; Rna15, small black diamond; CFI, large black diamond; RNAPII, grey shaded oval. doi:10.1371/journal.pone.0003273.g006 PLoS ONE | www.plosone.org 8 September 2008 | Volume 3 | Issue 9 | e3273